CN103877572A - Method for preparing porcine parvovirus disease inactivated vaccine - Google Patents
Method for preparing porcine parvovirus disease inactivated vaccine Download PDFInfo
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Abstract
The invention discloses a method for preparing a porcine parvovirus disease inactivated vaccine. The method comprises the following steps: (1) culturing cells: inoculating the cells to a micro-carrier of a cell culture bag, adding a cell medium, and shaking the cell culture bag to culture the cells; (2) proliferating a virus liquid: settling the micro-carrier, washing the cultured cells, inoculating a porcine parvovirus seed, and adding a virus proliferation culture base to continuously culture the cells so as to obtain the virus liquid; (3) inactivating the virus liquid, and emulsifying to obtain the inactivated vaccine. According to the method, each process parameter of ST cell culture in a wave bioreactor is optimized, the ST cell culturing efficiency is effectively improved, and the productivity efficiency and immune protection efficacy of the porcine parvovirus disease inactivated vaccine can be remarkably improved finally. Immune protective efficacy tests prove that the inactivated vaccine has an excellent immune protective effect on a pig, and the immune protective efficacy on the pig is remarkably superior to that of an inactivated vaccine prepared by a traditional spinner bottle.
Description
Technical field
The present invention relates to a kind of production method of porcine parvovirus inactivated vaccines, relate in particular to a kind of method of the WAVE of utilization wave bioreactor production porcine parvovirus inactivated vaccines, belong to the production field of porcine parvovirus inactivated vaccines.
Background technology
Pig parvoviral (Porcine Parvovirus) causes one of pig breeding dysfunction major reason.Main manifestations is that the negative multiparity sow of first farrowing sow and serum antibody is miscarried, stillbirth and fetus mummification, and sow does not show clinical symptoms conventionally.Primary disease is worldwide distribution, runs rampant widely in each pig farm.China, since nineteen eighty-three report primary disease, has approximately had 80% pig farm to exist PPV to infect, thus serious impact the development of the cause of raising pigs, develop a kind of primary disease of vaccine prevention safely and effectively for this reason and there is important practice significance.
Be porcine parvovirus inactivated vaccines at the vaccine of Chinese extensive use at present, what the production of vaccine adopted is traditional rolling bottle technique, and this technique has been used many decades in China's vaccine industry, and its operating technology is relatively simple and ripe.But each rolling bottle is all cell culture units independently, quality, viral yield and the titre of every bottle of cell is all different, cause between vaccine batch difference large, and operation labor intensity is large, production efficiency is low, the shortcomings such as the high endotoxin that recessive pollution causes, are more and more not suitable with the requirement of current vaccine large-scale production.
WAVE wave bioreactor is to adopt disposal type production technology to carry out a kind of novel reactor of cell culture.Its working method is, cell is inoculated in droppable cell culture bags, and the culture bag of inoculating cell is fixed on shake in some way on pallet, and the accurate shake of controlling has guaranteed effective mixing and the oxygen transfer efficiency of cultivating system.The present invention adopts WAVE wave bioreactor, and major advantage has 1) exempt bioreactor cleaning, sterilization and relevant authentication thereof.2) sealing culture systems, without fixing pipeline, simplifies cell culture Factory Building, shortens reactor and installs and produce the production interchange time.3) cell culture bags Cellbag is placed on the shake platform of particular design, and the shake of platform produces wave in culture fluid provides culture to mix and oxygen transmission, produces a perfect environment that is suitable for Growth of Cells.
While adopting WAVE wave bioreactor to produce porcine parvovirus inactivated vaccines; add the working conditions such as content, the cell inoculum density of microcarrier, the shake parameter of cell culture bags to have the impact of highly significant for the production efficiency of porcine parvovirus inactivated vaccines and the height of immune protection effectiveness etc., in production practices, need these conditions to be optimized production efficiency and the immune protection effectiveness that could effectively improve porcine parvovirus inactivated vaccines.
Summary of the invention
The object of this invention is to provide a kind of WAVE of utilization wave bioreactor and produce the method for porcine parvovirus inactivated vaccines; the parameters such as the content of the method to microcarrier, cell inoculum density, cell culture condition are optimized, and have effectively promoted production efficiency and the immune protection effectiveness of porcine parvovirus inactivated vaccines.
Above-mentioned purpose of the present invention is achieved through the following technical solutions:
Utilize WAVE wave bioreactor to produce a method for porcine parvovirus inactivated vaccines, comprising: the cultivation of (1) cell: ST cell is inoculated on the microcarrier in cell culture bags, adds cell culture medium, shake cell culture bags cultured cell; (2) propagation of virus liquid: sedimentation microcarrier, washing cultured cells, Pigs Inoculated parvovirus seed culture of viruses, adds virus multiplication culture medium culturing cell, results virus liquid; (3) by after virus liquid deactivation, emulsifying, obtains porcine parvovirus inactivated vaccines; Wherein, described shake condition is that 7 ° of pendulum angles, swing speed are 12rpm.
While adopting WAVE wave bioreactor culture ST cell, for cell, the absorption on microcarrier and Growth of Cells have the impact of highly significant to wave and culture parameter, in order to screen optimum wave and culture parameter with Promote cell's growth to greatest extent, the parameter such as pendulum angle and swing speed of the present invention during to wave and culture is optimized investigation, observes the impact that different condition of culture brings for the growth of cell.Found through experiments, in the time that the cell in cell culture bags carries out wave and culture, pendulum angle and swing speed have the impact of highly significant for the growth of cell; The present invention finally finds, the condition of culture in the time of wave and culture is that 7 ° of pendulum angles, swing speed are the growth that 12rpm can promote cell the most significantly, and the Growth of Cells speed under this condition of culture is significantly higher than other condition of culture.
The impact of cell inoculum density and microcarrier content cell growth is closely related, will guarantee the proportionate relationship of suitable cell density and carrier when wherein important mark is inoculation.Inoculate at every gram of microcarrier under the prerequisite of identical ST cell number, ST Growth of Cells situation when the present invention investigates Cytodex1 content and is 1g/L, 2g/L, 3g/L, 4g/L, 5g/L, 6g/L, 7g/L, 8g/L, is used for determining suitable carrier consumption.Can find out from experimental result, along with the increase of microcarrier content, ST cell density can increase to some extent, but ST cells expanded decreases.When microcarrier content is 3g/L, ST Growth of Cells is very fast, and when incubation time is 5 days, ST cell density has reached 2.05 × 10
7g/L, the cell density of the microcarrier higher than other of highly significant; In addition,, when microcarrier content is during higher than 3g/L, gathering way of ST Growth of Cells is not remarkable.Therefore,, when the present invention cultivates ST cell in cell culture bags, the content of the microcarrier adding is preferably 3g/L.Therefore consider, this experiment microcarrier content preferably adopts 3g/L.
Determining on the basis of microcarrier optimum consumption, the microcarrier consumption that the present invention is 3g/L, respectively with different inoculum density inoculating cells, every day sample analysis, to screen optimum cell inoculum density.From experimental result, when microcarrier content equates, different inoculum densities is larger on the impact of ST Growth of Cells.Different inoculum densities is larger on the impact of ST Growth of Cells.
The inventive method has been optimized each technological parameter of wave bioreactor culture ST cell, has effectively promoted the culture efficiency of ST cell, has finally significantly improved prouctiveness and the immune protection effectiveness of porcine parvovirus inactivated vaccines; Immune protection effectiveness experiment confirmation, the prepared inactivated vaccine of the inventive method has definite immune protection effectiveness for animal; The inventive method can be used for large-scale industrial production porcine parvovirus inactivated vaccines.
The specific embodiment
Further describe the present invention below in conjunction with specific embodiments, advantage and disadvantage of the present invention will be more clear along with description.But these embodiment are only exemplary, scope of the present invention are not formed to any restriction.It will be understood by those skilled in the art that lower without departing from the spirit and scope of the present invention and can the details of technical solution of the present invention and form be modified or be replaced, but these modifications and replacement all fall within the scope of protection of the present invention.
Biomaterial and instrument
1.1, bioreactor: the U.S. WAVE of Wave Biotech company wave bioreactor.
1.2, microcarrier: Cytodex-1(is purchased from Medical Group life sciences portion of General Electric (U.S.A.)).
1.3, pig parvoviral (CP-99 strain): purchased from Wuhan Chopper Biology Co., Ltd.; The strain that the deposit number obtaining from Chinese microorganism strain preservation administration committee's common micro-organisms center is CGMCCNo.6091 also can be applicable to the present invention.
Embodiment 1 utilizes WAVE wave bioreactor to produce porcine parvovirus inactivated vaccines
1. the cultivation of cell
1.1 microcarrier processing: take microcarrier by the final volume of cultivating appropriate, use without Ca
2+, Mg
2+-PBS soaking at room temperature is spent the night, and discards PBS, then uses without Ca
2+, Mg
2+-PBS washes once, discards, and finally adds without Ca
2+, Mg
2+-PBS autoclaving.115℃、10psi、15min。
1.2 cell recoveries are cultivated: the ST cell of recovering from liquid nitrogen container with flask culture, and condition of culture comprises: 37 ℃ of pH value 7.2, temperature; Cultivate 48-72h, while forming good cell monolayer, carry out microcarrier suspension culture for continuing to go down to posterity or be inoculated in bioreactor; The culture medium using in this process is MEM, and serum is hyclone, and use amount is 8%.
1.3 microcarriers are cultivated: prepare cell suspension with EDTA-pancreatin cell dissociation buffer, after cell counting, press 4 × 10
5the density of individual/mL is inoculated in cell culture bags cultivates.The method parameter of cultivating is: microcarrier concentration is 3g/L, DO value 50%, temperature 37 ℃ ﹑ pendulum angles 7,12 times/min of swing speed.In cultivation process, monitor the consumption of glucose in cell culture bags and the generation of lactic acid and ammonia, simultaneously on different nodes cell counting (cell counting on Cytodex1 is first used PBS rinsing 2 times, through the citric acid solution dyeing of 0.1% crystal violet, with blood counting chamber count fine karyon), when the density of cell reaches 1 × 10
6-2 × 10
6individual/to start perfusion when mL, according to the consumption of the density of cell, glucose to pour into the speed of 0.7-2 working volume every day, to maintain the generation of cell.
2. the propagation of virus liquid
Sedimentation microcarrier in the time that cell culture arrives the 4th day, discharge liquid in cell culture bags, add PBS washed cell, repeated washing 3 times, add viral maintenance medium Pigs Inoculated parvovirus, wherein virus inoculation dosage is 3%, viral value-added culture medium is the MEM containing 1% serum, pH value 7.4,35 ℃ of temperature.After connecing poison, get at regular intervals the microcarrier in bioreactor, by microscope observing cell pathological changes situation, and detect sample TCID50 and blood clotting valency, when the cell major part on microcarrier comes off, stop cultivating results virus liquid, in-20 ℃ of freeze thawing three times, obtain pig parvoviral stock solution.
The mensuration of 2.1 viral levels
Utilize TCID50 method to carry out the mensuration of viral level.When virus TCID50 measures, pig parvoviral liquid cultivation being obtained with MEM culture fluid is done continuous 10 times of dilutions, and 10
-1, 10
-210
-8, each dilution factor is got 100 μ L and is added in the hole of 96 porocyte culture plates, adds subsequently the ST cell suspension disperseing through trypsin-EDTA digestion, and every hole 100 μ L(cell contents are with 3 × 10
5/ mL is advisable left and right), each dilution factor is done 8 repetitions, and establishes normal cell and cultivate contrast, puts 5%CO
2in incubator, 37 ℃ of cultivations, observation of cell pathological changes and contrast day by day, observes 2～4 altogether, and records cytopathic hole count, calculates viral TCID50 according to Reed-Muench method.With identical method, the pig parvoviral stock solution with spinner culture is carried out to TCID50 mensuration simultaneously.With this as a control group.
Experimental result is in table 1; Experimental result shows, utilizes the pig parvoviral stock solution viral level of bioreactor culture to be not less than the pig parvoviral stock solution viral level of spinner culture.Utilize as can be seen here the virus of bioreactor culture will obviously be better than the virus of spinner culture.
Table 1 pig parvoviral stock solution viral level
2.2 viral hemoagglutination valencys are measured
Get respectively the toxic cell culture fluid of reactor and spinner culture and measure the hemagglutination activity to guinea-pig red blood cell, carry out with 96 hole micro plates (U-shaped), determined antigen is diluted to different multiples with pH7.2PBS, add 0.6% guinea-pig red blood cell suspension, shake up, put room temperature 60min, result of determination in the time that the erythrocyte in control wells is remarkable button-type, so that the high dilution of the complete coagulation of erythrocyte is as judging terminal.That detects the results are shown in Table 2.Shown by result, the pig parvoviral stock solution of utilizing bioreactor culture to the hemagglutination activity of guinea-pig red blood cell will be far away higher than the pig parvoviral stock solution of spinner culture the hemagglutination activity to guinea-pig red blood cell.Utilize as can be seen here the virus of bioreactor culture will obviously be better than the virus of spinner culture.
The hemagglutination test result of table 2 virus
Vaccine is made in 3 virus liquid deactivations and emulsifying: results divinyl imines (BEI) deactivation for virus liquid, 0.02%, 30 ℃ of deactivation 72h of BEI final concentration.Pig parvoviral liquid after deactivation adds conventional oil adjuvant, and stirring and evenly mixing, makes oil emulsion vaccine.
The optimization experiment of experimental example 1 microcarrier content
The impact of cell inoculum density and microcarrier content cell growth is closely related, will guarantee the proportionate relationship of suitable cell density and carrier when wherein important mark is inoculation.
Inoculate at every gram of microcarrier under the prerequisite of identical ST cell number, when investigation Cytodex1 content is 1g/L, 2g/L, 3g/L, 4g/L, 5g/L, 6g/L, 7g/L, 8g/L, ST Growth of Cells situation, is used for determining suitable carrier consumption.
As can be seen from Table 3, along with the increase of microcarrier content, ST cell density can increase to some extent, but ST cells expanded decreases.When microcarrier content is 3g/L, ST Growth of Cells is very fast, and when incubation time is 5 days, ST cell density has reached 1.85 × 10
7g/L, the cell density of the microcarrier higher than other of highly significant; In addition,, when microcarrier content is during higher than 3g/L, gathering way of ST Growth of Cells is not remarkable.Therefore,, when the present invention cultivates ST cell in cell culture bags, the content of the microcarrier adding is preferably 3g/L.Therefore consider, this experiment microcarrier content adopts 3g/L.
The different microcarrier content of table 3 Growth of Cells situation (unit/mL)
The screening experiment of experimental example 2ST cell inoculum density
When the consumption of microcarrier is 3g/L, respectively with 5 × 10
4, 1 × 10
5, 2 × 10
5, 3 × 10
5, 4 × 10
5, 5 × 10
5, 6 × 10
5the inoculum density inoculation ST cell of individual/mL, every day sample analysis, Growth of Cells situation is as shown in table 4.
As can be seen from Table 4, when microcarrier content equates, different inoculum densities is larger on the impact of ST Growth of Cells.With 5 × 10
4, 1 × 10
5, 2 × 10
5, 3 × 10
5when individual/mL inoculates, inoculum density is low, inoculates latter 5 days still in slow growth, does not enter the distinguishing mark of stable growth phase; With 4 × 10
5, 5 × 10
5, 6 × 10
5when individual/mL inoculates, inoculum density is high, and ST cell enters the stable growth phase in the 2nd day after inoculation; From the data of table 4, along with increasing of cell inoculum density, the increase of ST Growth of Cells speed does not increase and keeps synchronizeing with cell inoculum density; When ST cell inoculum density is 4 × 10
5individual/when mL, the speed of growth of ST cell after the 4th day and the 5th day will be apparently higher than the speed of growth of other inoculum density, and therefore, the present invention preferably adopts 4 × 10
5the inoculum concentration inoculation ST cell of individual/mL.
Table 4 different vaccination density Growth of Cells situation (unit/mL)
Experimental example 3 shakes the optimization experiment of condition
Content with 3g/L adds microcarrier, with 4 × 10
5individual/mL density inoculation ST cell, adds cell culture medium to cultivate, and pendulum angle is respectively 5 °, 6 °, 7 °, 8 °, 9 °; Swing speed is respectively 12rpm, 14rpm, 16rpm, 18rpm, and cell absorption and growing state on microcarrier are observed in sampling every day, investigate the impact of shake condition on cell culture.
Experimental result is in table 5.As can be seen from Table 5, whole cultivation process, shake condition is larger to the Effects of Density of cell.From experimental result, in the time that shake condition is 7 ° of pendulum angles, swing speed 12rpm, under this shake condition the speed of growth of cell will be far away higher than other shake condition (significant difference).Therefore, shake condition optimization of the present invention is 7 ° of pendulum angles, swing speed 12rpm.
Growth of Cells situation under the different shake conditions of table 5 (unit: individual/mL)
| 0(days) | 1(days) | 2(days) | 3(days) | 4(days) | 5(days) | |
| 6°、12 ｒｐｍ | 4×10 5 | 5.2×10 5 | 4.16×10 6 | 9.52×10 6 | 1.17×10 7 | 1.77×10 7 |
| 6°、14 ｒｐｍ | 4×10 5 | 6.8×10 5 | 4.61×10 6 | 9.82×10 6 | 1.23×10 7 | 1.82×10 7 |
| 6°、16 ｒｐｍ | 4×10 5 | 6.3×10 5 | 4.48×10 6 | 9.71×10 6 | 1.09×10 7 | 1.79×10 7 |
| 6°、18 ｒｐｍ | 4×10 5 | 5.8×10 5 | 4.39×10 6 | 9.62×10 6 | 1.14×10 7 | 1.68×10 7 |
| 7°、12 ｒｐｍ | 4×10 5 | 7.3×10 5 | 5.21×10 6 | 1.68×10 7 | 2.08×10 7 | 3.23×10 7 |
| 7°、14ｒｐｍ | 4×10 5 | 6.92×10 5 | 4.58×10 6 | 9.79×10 6 | 1.22×10 7 | 1.92×10 7 |
| 7°、16 ｒｐｍ | 4×10 5 | 5.7×10 5 | 4.21×10 6 | 9.08×10 6 | 1.02×10 7 | 1.21×10 7 |
| 7°、18 ｒｐｍ | 4×10 5 | 5.5×10 5 | 4.27×10 6 | 9.62×10 6 | 1.12×10 7 | 1.71×10 7 |
| 8°、12 ｒｐｍ | 4×10 5 | 5.9×10 5 | 4.44×10 6 | 9.75×10 6 | 1.16×10 7 | 1.84×10 7 |
| 8°、14ｒｐｍ | 4×10 5 | 6.6×10 5 | 4.13×10 6 | 9.73×10 6 | 1.18×10 7 | 1.16×10 7 |
| 8°、16 ｒｐｍ | 4×10 5 | 6.8×10 5 | 4.51×10 6 | 9.81×10 6 | 1.26×10 7 | 1.87×10 7 |
| 8°、18 ｒｐｍ | 4×10 5 | 6.3×10 5 | 4.30×10 6 | 9.59×10 6 | 1.17×10 7 | 1.78×10 7 |
| 9°、12 ｒｐｍ | 4×10 5 | 6.46×10 5 | 4.30×10 6 | 9.61×10 6 | 1.12×10 7 | 1.72×10 7 |
| 9°、14 ｒｐｍ | 4×10 5 | 6.69×10 5 | 4.30×10 6 | 9.82×10 6 | 1.23×10 7 | 1.81×10 7 |
| 9° 16ｒｐｍ | 4×10 5 | 6.51×10 5 | 4.30×10 6 | 9.78×10 6 | 1.15×10 7 | 1.69×10 7 |
| 9°、18ｒｐｍ | 4×10 5 | 6.58×10 5 | 4.30×10 6 | 9.69×10 6 | 1.06×10 7 | 1.57×10 7 |
The safety experiment of experimental example 4 vaccines
One, the inactivated vaccine of preparing for examination vaccine: embodiment 1.
Two, experimental technique and result
1. the safety test of a single dose inoculation: 10 PPV HI negative antibody pigs of vaccination, 2mL/ head, intramuscular injection, observes 21.
2. single dose repeated inoculation safety test: 10 PPV HI negative antibody pigs of vaccination, inoculate for the first time 2mL/ head, 2 weeks, interval, intramuscular inoculation is with batch vaccine again, and 2mL/ head, observes 21.
3. the safety test of an overdose inoculation: 10 PPV HI negative antibody pigs of vaccination, 10mL/ head, intramuscular injection, observes 21.
4. the safety testing of farrowing sow: select PPV HI negative antibody, gestation 10 sows of 1.5～2 months, every inoculation 10mL vaccine, intramuscular injection, observes to farrowing.
5. sow reproductive function impact test: 10 of the first farrowing sows of selection PPV HI negative antibody, vaccination about 6 monthly ages, 10mL/ head, breeding about 1～1.5 month in interval, observes whole trimester of pregnancy, until farrowing.
6. the safety test of white mice: vaccination 1 nest 2～4 age in days white mice (at least 5), only, subcutaneous injection, observes 10 0.1mL/.
Above result of the test, has no after immunity inoculation reproductive function is had to any impact, and body temperature, feed, breeding, gestation, farrowing are all normal.Experiment results proved, inactivated vaccine safety of the present invention is good.The immunogenicity comparative experiments of experimental example 5 vaccines
One, experiment material
1, the inactivated vaccine of preparing for examination vaccine: embodiment 1.
2, control vaccine: the rolling bottle Seedling that adopts existing conventional rolling bottle method to prepare.
Two, experimental technique and result
To supply examination vaccine and rolling bottle vaccine to inoculate respectively the test pig of PPV HI antibody titer < 4Log2, each 10, to observe 14, blood sampling and attack are strong malicious.After counteracting toxic substances 6 days, blood sampling, separation of serum, measures serum HI antibody, the results are shown in Table 6.As can be seen from the results, vaccination pig was in latter 14 days of inoculation, and reactor vaccine and rolling bottle vaccine all can produce antibody response reaction, but the HI antibody titer that reactor vaccine produces is compared with the height of rolling bottle vaccine, attack after strong poison, in serum, HI tires and does not increase, and shows that strong poison do not breed in pig body.
Table 6 Pigs Inoculated challenge test result
2. vaccine is to in-pig protection test result
Select the first farrowing sow of PPV HI antibody titer < 4Log2, inoculation is for examination vaccine and rolling bottle vaccine respectively, breeding in 1～1.5 month after vaccination, after pregnancy, take a blood sample and attack PPV poison by force, within the 6th day after counteracting toxic substances, take a blood sample and cut open and kill, separation of serum is measured HI antibody titer, gather the tissue sample of stillborn fetus, isolated viral, check result is in table 7.Can be found out by result, will slightly be better than rolling bottle vaccine for examination vaccine to the protection effect of in-pig.
Table 7 vaccine is to in-pig protection test comparison
3. vaccine is to the pig comparison of immunity time
By rolling bottle vaccine, inoculate respectively 10 of the health pig of HI antibody titer < 4Log2 for examination vaccine, separately establish 10 contrast health pig, and different time blood sampling after vaccination, measure HI antibody titer in serum, the results are shown in Table 8.
HI antibody titer measurement result after table 8 vaccination
From table 8, two kinds of vaccinated pigs all produce antibody, from producing antibody horizontal height, and will be higher than rolling bottle vaccine for examination vaccine; From antibody Fluctuation, the antibody persistent period that the antibody persistent period producing for examination vaccine produces than rolling bottle vaccine is long, shown by above data, the porcine parvovirus inactivated vaccines that utilizes WAVE wave bioreactor to produce is better than the vaccine of producing with rolling bottle.
Claims (8)
1. a preparation method for porcine parvovirus inactivated vaccines, comprising: the cultivation of (1) cell: seed cells on the microcarrier of cell culture bags, add cell culture medium, shake cell culture bags cultured cell; (2) propagation of virus liquid: sedimentation microcarrier, washing cultured cells; Pigs Inoculated parvovirus seed culture of viruses, adds virus multiplication culture medium to continue cultured cell, results virus liquid; (3) by after virus liquid deactivation, emulsifying, obtains porcine parvovirus inactivated vaccines; It is characterized in that: the shake parameter described in step (1) is that 7 ° of pendulum angles, swing speed are 12rpm.
2. it is characterized in that in accordance with the method for claim 1: described cell is ST cell.
3. it is characterized in that in accordance with the method for claim 1: described microcarrier is Cytodex-1.
4. it is characterized in that in accordance with the method for claim 1: the content of microcarrier in cell culture bags is 3g/L.
5. it is characterized in that in accordance with the method for claim 1: by cell according to 4 × 10
5the inoculum concentration of individual/mL is inoculated on the microcarrier of cell culture bags.
6. by the claim 1-5 porcine parvovirus inactivated vaccines that method prepares described in any one.
7. porcine parvovirus inactivated vaccines claimed in claim 7 is caused the purposes in disease medicament in preparation prevention or treatment by pig parvoviral.
8. prevention or treatment, by a pharmaceutical composition for disease that pig parvoviral causes, is characterized in that: the porcine parvovirus inactivated vaccines claimed in claim 6 that contains prevention or the upper effective dose for the treatment of.
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Application publication date: 20140625 |