CN102743748A - Method for preparing swine fever live vaccine by using oscillatory type bioreactor - Google Patents
Method for preparing swine fever live vaccine by using oscillatory type bioreactor Download PDFInfo
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Abstract
The invention provides a preparation method of swine fever live vaccine, which is characterized in that cells used for sprout is inoculated to an oscillatory type bioreactor containing a nutrient solution and a microcarrier, and the cells are attached on the microcarrier. When the cell density reach the 108/g microcarrier, the hog cholera lapinized low virulent strain is inoculated, and then the virus is breed. A virus liquid is obtained, clarified and filtered, and then a freeze-drying protective agent is added, fully mixed and quantifiably packed, performed freeze-drying to obtain the swine fever live vaccine. The method has the advantages of fast cell growth, high concentration, continuous culture and gains, high virus output and simple production technology. The method for swine fever live vaccine is better then a traditional method for swine fever live vaccine, and has good application prospect.
Description
Technical field
The present invention relates to the vaccine production field, particularly, relate to the method that adopts the oscillatory type bioreactor to prepare live vaccines of hog cholera.
Background technology
(classical swine fever CSF) betides the Ohio of the U.S. in 1833 to swine fever the earliest, just claims hog cholera, and 1903 are proved to be pathogen is a kind of virus, and Europe then is referred to as classic swine fever.There is the research report aspect the immune serum nineteen twenty-five in the Southeast China University academy of agricultural sciences the earliest in China.Swine fever is one of the most serious disease of pig in the world wide, thereby is classified as the category-A infectious disease by OIE (World Organization for Animal Health), and China also classifies it as type of infectious disease.Its cause of disease is swine fever virus (CSFV), belongs to flaviviridae pestivirus member.With hemorrhage and heating is principal character, is a kind of infectious disease very harmful to pig.
The swine Fever Vaccine of current use all is with rabbitization attenuated vaccine (HCLV, promptly famous C strain, or strain in China), is divided into according to the difference of production technology: live vaccines of hog cholera (cell source), i.e. swine fever cell vaccine; Live vaccines of hog cholera (rabbit source), promptly swine fever is organized Seedling, and it still is that newborn rabbit is divided into swine fever spleen pouring Seedling and swine fever breast rabbit Seedling according to producing with adult rabbit again that swine fever is organized Seedling.The outstanding advantage of the weak poison of universally acknowledged Chinese C strain rabbitization is; Pig with its inoculation does not produce viremia and encephalitis pathological changes; Sow and the suckling pig of immunity inoculation any period of pregnancy do not influence rate of fertilization and survival rate safely, do not cause miscarriage, stillborn fetus; Can be used for setting up kind of a swinery (Tesmer etc., 1973) safely.Commonly used to also have Japanese GPE strain, French Thiverval strain etc. all be the CSFV vaccine strain of extensive use, the general equal semelincident immunization that provides.
Its deficiency of technology that tradition rolling bottle legal system is equipped with live vaccines of hog cholera is the danger that exists external source to pollute, and it is lower to produce malicious titre; Effectively working cell quantity is lower in the unit volume; Inefficiency, batch differences is big, poor stability.Therefore developing the novel method for preparing swine Fever Vaccine is to improve viral yield and the key of practicing thrift cost.
Summary of the invention
The object of the present invention is to provide stability and high efficiency to prepare the method for live vaccines of hog cholera, to overcome the deficiency of prior art.
The present invention provides a kind of method that adopts the oscillatory type bioreactor to prepare live vaccines of hog cholera, comprises the following steps:
(1) cultivates seedling and use cell
With seedling with cell inoculation in the bioreactor jar that contains cell culture fluid and microcarrier; Start cell absorption program, behind oscillatory type absorption 3～4h or the cell adsorption rate reach 80% when above, be converted into the cell culture program; Carry out the oscillatory type cell culture, treat that cell grows to 1.5 * 10
8～3 * 10
8Individual/as during the g microcarrier, to stop to cultivate;
(2) breeding seedling venom
The swine fever virus suspension inoculation in step (1) cultured cells, behind inversion absorption 0.5～1h, is carried out the oscillatory type Virus culture, and per 1～2d gathers in the crops viral liquid, and in time changes and keep liquid, and harvesting frequency is 15～20 times, gathers the viral liquid of results;
(3) will add freeze drying protectant through the viral liquid clarification filtration that is up to the standards, fully quantitatively packing behind the mixing, lyophilizing promptly obtains live vaccines of hog cholera.
The used bioreactor of the inventive method is a BelloCell type oscillatory type cell microcarrier suspension culture bioreactor.This bioreactor mainly is made up of main body board, controller and culture bottle.Work process is following: the main body board can move up and down the thin plate platform by step motor driven.When platform rose, the culture medium in the BelloCell-500 in the flexible bottle in below was squeezed to body, and makes carrier be infiltrated in the culture medium, lets cell carry out the exchange of required nutrient and garbage; When platform descended, culture medium then was back to the flexible bottle in below once more, and carrier is exposed in the air, let the oxygen quick exchange, formed the good cell culture environment.
The interior BioNoc II microcarrier density of bioreactor jar is 11g/L in the inventive method step (1), and inserting cell concentration is 1 * 10
7/ g～1 * 10
8/ g microcarrier.
The microcarrier that uses is 865 ± 5% as porous adhesive-bonded fabric, microcarrier quantity, is about 5.5g, accounts for the space of 10ml, can provide cell growth area to reach 2400cm approximately
2/ g.Said microcarrier is netted and/or lamellar.
Said step (1) oscillatory type cell absorption program parameter is: cell culture fluid is 1.0mm/s through the rate of climb of microcarrier, and the top stops 25s, and decrease speed is 1.0mm/s, and the bottom stops 0s; Oscillatory type cell culture program parameter is: cell culture fluid is 1.5mm/s through the rate of climb of microcarrier, and the top stops 0s, and decrease speed is 1.5mm/s, and the bottom stops 60s.
Inoculation 10%～20% hog cholera lapinised virus strain cell seed culture of viruses or 1%～2% hog cholera lapinised virus strain spleen seed culture of viruses in the step of the present invention (2), said percentage ratio is the volume ratio of seed culture of viruses and virus-culturing fluid.
Said hog cholera lapinised virus strain cell seed culture of viruses virus titer is: every 1ml contains virus >=100000 a rabbit infective dose.
Said hog cholera lapinised virus strain spleen seed culture of viruses virus titer is: every 1ml contains virus >=100000 a rabbit infective dose.
Wherein step (2) oscillatory type Virus culture parameter is virus-culturing fluid through microcarrier the rate of climb is 1.0mm/s, and the top stops 10s; Virus-culturing fluid is through the decrease speed 1.0mm/s of microcarrier, and the bottom stops 30s.
Should be appreciated that those skilled in the art can carry out the adjustment of reaction vessel control parameter according to practical condition.
The every 1ml of viral liquid that obtains after step of the present invention (2) Virus culture contains virus >=1000000 a rabbit infective dose.
Seedling of the present invention uses cell to be PK15 cell line.
Cell culture fluid of the present invention is the DMEM that contains 5%～8% hyclone, and virus-culturing fluid, to keep liquid be the MEM that contains 1～2% hyclone, and pH value is 7.0～7.4.
The condition of cell culture of the present invention and Virus culture is: temperature is 36.5～37 ℃, CO
2Concentration 1%～5%.
Freeze drying protectant described in the inventive method step (3) is 5% sucrose skim milk.
The invention provides the application of said method in the preparation live vaccines of hog cholera.
The invention provides the live vaccines of hog cholera that method for preparing obtains.
The invention provides a kind of method that adopts the oscillatory type bioreactor to prepare live vaccines of hog cholera; Its beneficial effect is 1) the swine fever virus poison valency produced of the inventive method is high; Rabbit body heat reaction content is than with about 10 times of the swine fever virus height of traditional rolling bottle explained hereafter; The vaccine that makes is carried out each item checks such as aseptic, mycoplasma, discriminating, exogenous virus, safety, effectiveness according to " People's Republic of China's veterinary drug allusion quotation (2010 editions) " method, all meet middle regulation; 2) use the swine fever virus technology that oscillatory type microcarrier reaction vessel is cultivated the PK15 cell; Microcarrier provides bigger cell attachment surface area, and the cell yield of unit volume culture fluid is high, and observation of cell is at the growing state of carrier surface at any time; High culture density PK15 cell can improve virus titer; Best biochemical condition when this method is monitored cell growth or virus breeding at any time, and every batch reaction device is produced the virus titer homogeneous of supernatant, culture medium utilization rate height; Virus results process is uncomplicated, little, the steady quality of vaccine batch differences; 3) adopt oscillatory type cell microcarrier suspension culture system of the present invention, compare with traditional suspension culture system, cell culture processes is simple, does not produce shearing force and bubble during cultivation, and the cell growing environment is good; 4) culture systems of the inventive method take up room little, and the operation labor intensity little, reduce pollution section, make it have remarkable economic efficiency.
Description of drawings
Fig. 1 is cell inoculation microphotograph of cell on the microcarrier in the time of 2 days, and the arrow indication is the cell on the microcarrier;
Fig. 2 is virus inoculation microphotograph of cell on the microcarrier in the time of 5 days, and the arrow indication is the cell on the microcarrier;
Fig. 3 is an oscillatory type microcarrier suspension culture bioreactor construction sketch map.
The specific embodiment
Following examples are used to explain the present invention, but are not used for limiting scope of the present invention.Under the situation that does not deviate from the present invention's spirit and essence, modification or replacement to the inventive method, step or condition are done all belong to scope of the present invention.
Employed bioreactor is BelloCell type bioreactor (the main engine bed BelloStage-3000 of U.S. match space company in the embodiment of the invention; Controller, tank body BelloCell-500), employed carrier is a BioNoc II porous adhesive-bonded fabric; Amount vector is 865 ± 5% carriers; Be about 5.5g, account for the space of 10ml, can provide cell growth area to reach 2400cm approximately
2/ g.
Employed bioreactor mainly is made up of main body board, controller and culture bottle among the embodiment.Work process is following: the main body board can move up and down the thin plate platform by step motor driven.When platform rose, the culture medium in the BelloCell-500 in the flexible bottle in below was squeezed to body, and makes carrier be infiltrated in the culture medium, lets cell carry out the exchange of required nutrient and garbage; When platform descended, culture medium then was back to the flexible bottle in below once more, and carrier is exposed in the air, let the oxygen quick exchange, formed the good cell culture environment.
If do not specialize the conventional means that used technological means is well known to those skilled in the art among the embodiment.
Embodiment 1 usefulness oscillatory type microcarrier suspension bioreactor large-scale production swine fever virus (1)
1, virus and cell strain
The virus that is used to make live vaccines of hog cholera is hog cholera lapinised virus strain (C strain); Derive from veterinary microorganism DSMZ of country of China Veterinery Drug Inspection Office; Through pathogenic test; This low virulent strain virus does not have pathogenic (this strain is identified according to the cell seed culture of viruses and met hog cholera lapinised virus strain seed culture of viruses standard fully, pig safety is had no side effect).With the porcine kidney cell line (PK15) that hog cholera lapinised virus strain (C strain) is had good sensitivity, use cell line as seedling.
2, method for preparing
2.1 seedling is with the cultivation of cell: with seedling with cell line PK15 cell inoculation in the tank body that contains 500mLDMEM fluid medium (comprising 8% hyclone (FBS), 100UI/mL penicillin, 100 μ g/mL streptomycin pH7.2) and 5.5g BioNoc II carrier; Cell initial inoculation amount is 1.25 * 10
7Individual cell/g microcarrier.In 37 ℃, 5%CO
2Under the culture environment, cell absorption 4h makes on its maximum ground absorption carrier.Cell absorption program parameter is: culture fluid is 1.0mm/s through the rate of climb of microcarrier, and the top stops 25s, and decrease speed is 1.0mm/s, and the bottom stops 0s; Behind the 4h, the cell adsorption rate reaches more than 80%, switches to the cell culture program, and inoculating cell is grown the most efficiently.The rate of climb that the cell culture program parameter is a culture fluid through microcarrier is 1.5mm/s, and the top stops 0s, and decrease speed is 1.5mm/s, and the bottom stops 60s.Cell inoculation in the time of 2 days on the microcarrier microphotograph of cell see Fig. 1.
2.2 the breeding of seedling venom: in 37 ℃, 5%CO
2Under the culture environment, cell was grown on carrier 4 days, and cell number reaches 3 * 10
8Individual cell/g microcarrier is inoculated in above-mentioned cell by 20%, 2% of virus-culturing fluid volume respectively separately with rabbitization low virulent strain cell seed culture of viruses or hog cholera lapinised virus strain spleen seed culture of viruses (every 1ml contains virus>=100000 a rabbit infective dose).With MEM is that (comprise 1%FBS, 100UI/mL penicillin, 100 μ g/mL streptomycins, pH7.2), behind the inversion absorption 1h, start the Virus culture program, promptly culture fluid is 1.0mm/s through the rate of climb of microcarrier to fluid medium, continues 10s; Culture fluid is 1.0mm/s through the decrease speed of microcarrier, continues 30s.In 37 ℃, 5%CO
2Continue to cultivate under the culture environment, change liquid every day, gather in the crops viral liquid once every day, gather in the crops 20 times continuously, and put-20 ℃ of preservations from the beginning in the 4th day that connects behind the poison.Virus inoculation in the time of 5 days on the microcarrier microphotograph of cell see Fig. 2.The employed oscillatory type microcarrier suspension culture of present embodiment bioreactor construction sketch map is seen Fig. 3.
Gather in the crops the check of viral liquid (semi-finished product): test for 42,49 pages according to " People's Republic of China's veterinary drug allusion quotation " (version in 2010) appendix, assay does not have antibacterial, mycete, mycoplasma growth.The cell venom has no side effect to pig safety, and the viral liquid of each time results is measured with rabbit respectively, and every 1ml contains virus >=1000000 a rabbit infective dose.
2.3 join Seedling, packing and lyophilizing:, be mixed in the same container, behind the clarification filtration through the viral liquid that is up to the standards; Add 5% sucrose skimmed milk stabilizing agent in 1: 1 ratio, fully shake up, quantitatively packing; Every part contains the cell venom and is no less than 0.015mL, makes finished product after the packing lyophilizing.
Product inspection: test according to " Chinese people republic veterinary drug allusion quotation " (version in 2010).
With swine fever cell toxicant, swine fever spleen poison is that the seedling seed culture of viruses uses said method to process 3 batches of vaccines respectively; The vaccine that totally 6 batches of vaccines, the vaccine numbering in swine fever cell toxicant source are respectively PK15-1-01, PK15-1-02, PK15-1-03 and swine fever spleen poison source is numbered PK15-1-11, PK15-1-12, PK15-1-13.
3, result
3.1 the inspection of semifinished product: prepare 6 batches of semi-finished product; Require to have carried out steriling test, mycoplasma check, mycoplasma check according to " Chinese people republic veterinary drug allusion quotation " (version in 2010); 6 batches of semi-finished product all do not detect antibacterial, mycete, mycoplasma and exogenous virus; 6 batches of semi-finished product viral levels reach 1,000,000 rabbit infective dose/mL, are that traditional rolling bottle culture process is produced (20 times of 50,000 rabbit infective doses/mL) of vaccine (cell source) semi-finished product standard a little less than the swine fever rabbitization.
3.2 product inspection
3.2.1 physical behavior check: 6 batches of vaccines of preparation, it is the spongy loose agglomerate of milky, is prone to break away from the bottle wall, add diluent after dissolving rapidly, free from extraneous odour.
3.2.2 steriling test: test for 42 pages according to " Chinese people republic veterinary drug allusion quotation " (version in 2010) appendix, made vaccine is asepsis growth.
3.2.3 mycoplasma check: test for 49 pages according to " Chinese people republic veterinary drug allusion quotation " (version in 2010) appendix, made vaccine is no mycoplasma and grows.
3.2.4 diagnostic test: vaccine is diluted to the viral suspension that every ml contains the minimal infecting dose (MID) of 100 rabbits with normal saline, fully mixes, put 15 ℃ of effects 60 minutes with the swine fever virus resistant specific serum of equivalent, during jolting 2～3 times.Establish the contrast of virus control and normal saline simultaneously.Neutralization is inoculated 2 of rabbits respectively after finishing, every rabbit ear vein injection 1.0ml, and temp measuring method and body temperature reaction normal are with the efficacy test item.The result of 6 batches of vaccine samples, except that thermal response appearred in positive controls, thermal response did not all appear in all the other groups in the 120h after inoculation.
3.2.5 exogenous virus check: test for 40 pages according to " Chinese people republic veterinary drug allusion quotation " (version in 2010) appendix, made vaccine is no exogenous virus pollution.
3.2.6 safety verification: with normal saline vaccine is diluted to 6 part/ml, 4 of the healthy susceptible pigs of every batch of vaccine difference intramuscular injection, every 5ml (containing 30 parts).Inoculation back thermometric is observed, and is undertaken by " Chinese people republic veterinary drug allusion quotation " (version in 2010) 72 pages.The result shows that each batch test pig body temperature is normal, and other signs are normal, judges that vaccine is qualified.
3.2.7 efficacy test: select method with test-free; Vaccine is diluted to variable concentrations with sterile saline; 2 of the rabbits of auricular vein injection body weight 1.5～3.0kg, every 1.0ml carries out according to " Chinese people republic veterinary drug allusion quotation " (version in 2010) 72 pages.Vaccine potency is up to the standards, and every part is contained 15000 rabbit infective doses, is 20 times of traditional live vaccines of hog cholera (cell source) vaccine potency standard, and the result sees table 1.
Table 1 rabbit efficacy test result
Annotate: ++ be the typing thermal response ,+be that slight fever is reacted ,-be reactionless.
3.8 residual moisture is measured: test for 38 pages according to " Chinese people republic veterinary drug allusion quotation " (version in 2010) appendix, made vaccine is all up to specification.
3.9 vacuum is measured: test for 48 pages according to " Chinese people republic veterinary drug allusion quotation " (version in 2010) appendix, made vaccine is all up to specification.
4 brief summaries
Live vaccines of hog cholera semi-finished product through the inventive method preparation, finished product be particularly outstanding aspect the effectiveness, is 20 times that traditional rolling bottle culture process is produced vaccine (cell source) semi-finished product a little less than the swine fever rabbitization, finished product viral level standard (effectiveness).
Embodiment 2 usefulness oscillatory type microcarrier suspension bioreactor large-scale production swine fever virus (2)
1, virus and cell strain
The virus that is used to make live vaccines of hog cholera is hog cholera lapinised virus strain (C strain); Derive from veterinary microorganism DSMZ of country of China Veterinery Drug Inspection Office; Through pathogenic test; This low virulent strain virus does not have pathogenic (this strain is identified according to the cell seed culture of viruses and met hog cholera lapinised virus strain seed culture of viruses standard fully, pig safety is had no side effect).With the porcine kidney cell line (PK15) that hog cholera lapinised virus strain (C strain) is had good sensitivity, use cell line as seedling.
2, method for preparing
2.1 seedling is with the cultivation of cell: with seedling with cell line PK15 cell inoculation in the tank body that contains 500mLDMEM fluid medium (comprising 5% hyclone (FBS), 100UI/mL penicillin, 100 μ g/mL streptomycin pH7.0) and 5.5g BioNoc II carrier; Cell initial inoculation amount is 1 * 10
7Individual cell/g microcarrier.In 37 ℃, 5%CO
2Under the culture environment, cell absorption 4h makes on its maximum ground absorption carrier.Cell absorption program parameter is: culture fluid is 1.0mm/s through the rate of climb of microcarrier, and the top stops 25s, and decrease speed is 1.0mm/s, and the bottom stops 0s; Behind the 4h, the cell adsorption rate reaches more than 80%, switches to the cell culture program, and inoculating cell is grown the most efficiently.The rate of climb that the cell culture program parameter is a culture fluid through microcarrier is 1.5mm/s, and the top stops 0s, and decrease speed is 1.5mm/s, and the bottom stops 60s.
2.2 the breeding of seedling venom: in 37 ℃, 5%CO
2Under the culture environment, cell was grown on carrier 3 days, and cell number reaches 1.5 * 10
8Individual cell/g microcarrier, with rabbitization low virulent strain cell seed culture of viruses or hog cholera lapinised virus strain spleen seed culture of viruses respectively separately (every 1ml contains virus>=100000 a rabbit infective dose) be inoculated in above-mentioned cell culture fluid by 10%, 1% of virus-culturing fluid volume.Be inoculated in above-mentioned cell.With MEM is that (comprise 2%FBS, 100UI/mL penicillin, 100 μ g/mL streptomycins, pH7.4), behind the inversion absorption 0.5h, start the Virus culture program, promptly culture fluid is 1.0mm/s through the rate of climb of microcarrier to fluid medium, continues 10s; Culture fluid is 1.0mm/s through the decrease speed of microcarrier, continues 30s.In 37 ℃, 1%CO
2Continue to cultivate under the culture environment, changed liquid once in per 2 days,, gathered in the crops viral liquid once in per 2 days, gather in the crops 15 times continuously, and put-20 ℃ of preservations from connecing the beginning in the 4th day behind the poison.
The check of the viral liquid (semi-finished product) of results: test for 42,49 pages according to " People's Republic of China's veterinary drug allusion quotation " (version in 2010) appendix, assay does not have antibacterial, mycete, mycoplasma growth.The cell venom has no side effect to pig safety, and the viral liquid of each time results is measured with rabbit respectively, and every 1ml contains virus >=1000000 a rabbit infective dose.
2.3 join Seedling, packing and lyophilizing:, be mixed in the same container, behind the clarification filtration through the viral liquid that is up to the standards; Add 5% sucrose skimmed milk stabilizing agent in 1: 1 ratio, fully shake up, quantitatively packing; Every part contains the cell venom and is no less than 0.015mL, makes finished product after the packing lyophilizing.
Product inspection: test according to " Chinese people republic veterinary drug allusion quotation " (version in 2010).
With swine fever cell toxicant, swine fever spleen poison is that the seedling seed culture of viruses uses said method to process 3 batches of vaccines respectively; The vaccine that totally 6 batches of vaccines, the vaccine numbering in swine fever cell toxicant source are respectively PK15-2-01, PK15-2-02, PK15-2-03 and swine fever spleen poison source is numbered PK15-2-11, PK15-2-12, PK15-2-13.
3, result
3.1 the inspection of semifinished product: prepare 6 batches of semi-finished product; Require to have carried out steriling test, mycoplasma check, mycoplasma check according to " Chinese people republic veterinary drug allusion quotation " (version in 2010); 6 batches of semi-finished product all do not detect antibacterial, mycete, mycoplasma and exogenous virus; 6 batches of semi-finished product viral levels reach 1,000,000 rabbit infective dose/mL, are that traditional rolling bottle culture process is produced (20 times of 50,000 rabbit infective doses/mL) of vaccine (cell source) semi-finished product standard a little less than the swine fever rabbitization.
3.2 product inspection
3.2.1 physical behavior check: 6 batches of vaccines of preparation, it is the spongy loose agglomerate of milky, is prone to break away from the bottle wall, add diluent after dissolving rapidly, free from extraneous odour.
3.2.2 steriling test: test for 42 pages according to " Chinese people republic veterinary drug allusion quotation " (version in 2010) appendix, made vaccine is asepsis growth.
3.2.3 mycoplasma check: test for 49 pages according to " Chinese people republic veterinary drug allusion quotation " (version in 2010) appendix, made vaccine is no mycoplasma and grows.
3.2.4 diagnostic test: vaccine is diluted to the viral suspension that every ml contains the minimal infecting dose (MID) of 100 rabbits with normal saline, fully mixes, put 10 ℃ of effects 60 minutes with the swine fever virus resistant specific serum of equivalent, during jolting 2-3 time.Establish the contrast of virus control and normal saline simultaneously.Neutralization is inoculated 2 of rabbits respectively after finishing, every rabbit ear vein injection 1.0ml, and temp measuring method and body temperature reaction normal are with the efficacy test item.The result of 6 batches of vaccine samples, except that thermal response appearred in positive controls, thermal response did not all appear in all the other groups in the 120h after inoculation.
3.2.5 exogenous virus check: test for 40 pages according to " Chinese people republic veterinary drug allusion quotation " (version in 2010) appendix, made vaccine is no exogenous virus pollution.
3.2.6 safety verification: with normal saline vaccine is diluted to 6 part/ml, 4 of the healthy susceptible pigs of every batch of vaccine difference intramuscular injection, every 5ml (containing 30 parts).Inoculation back thermometric is observed, and is undertaken by " Chinese people republic veterinary drug allusion quotation " (version in 2010) 72 pages.The result shows that each batch test pig body temperature is normal, and other signs are normal, judges that vaccine is qualified.
3.2.7 efficacy test: select method with test-free; Vaccine is diluted to variable concentrations with sterile saline; 2 of the rabbits of auricular vein injection body weight 1.5-3.0kg, every 1.0ml carries out according to " Chinese people republic veterinary drug allusion quotation " (version in 2010) 72 pages.Vaccine potency is up to the standards, and every part is contained 15000 rabbit infective doses, is 20 times of traditional live vaccines of hog cholera (cell source) vaccine potency standard, and the result sees table 2.
Table 2 rabbit efficacy test result
Annotate: ++ be the typing thermal response ,+be that slight fever is reacted ,-be reactionless.
3.8 residual moisture is measured: test for 38 pages according to " Chinese people republic veterinary drug allusion quotation " (version in 2010) appendix, made vaccine is all up to specification.
3.9 vacuum is measured: test for 48 pages according to " Chinese people republic veterinary drug allusion quotation " (version in 2010) appendix, made vaccine is all up to specification.
4 brief summaries
Live vaccines of hog cholera semi-finished product through the inventive method preparation, finished product be particularly outstanding aspect the effectiveness, is 20 times that traditional rolling bottle culture process is produced vaccine (cell source) semi-finished product a little less than the swine fever rabbitization, finished product viral level standard (effectiveness).
The effect comparative test of the swine fever that embodiment 3 the present invention make malicious vaccine alive and like product
1, material
1.1 the swine fever that vaccine: embodiment 1 makes 3 batches of malicious vaccines (cell toxicant source) alive, lot number is PK15-1-01, PK15-1-02, PK15-1-03.1 batch of the live vaccines of hog cholera that makes with traditional rolling bottle method: ZP-01.
1.2 experimental animal: select the feed lot that meets government test animal standard for use, cleaning level rabbit (be placed on before the inoculation in the container of cleaning, put in the 20-30 ℃ of clean environment, morning and afternoon every day, each was observed once), rabbit to be inoculated does not have abnormal symptom.
Select for use the feed lot that meets government test animal standard or the supply of fixed point pig farm the healthy susceptible wean of no hog cholera antibody pig (observe 5-7 day before annotating Seedling, morning and afternoon every day each thermometric once, select body temperature, spirit, the normal person of appetite and use).
2, method
2.1 physical behavior check: the physical behavior to 4 batches of vaccines of preparation is estimated.
2.2 steriling test: test for 42 pages according to " Chinese people republic veterinary drug allusion quotation " (version in 2010) appendix.
2.3 mycoplasma check: test for 49 pages according to " Chinese people republic veterinary drug allusion quotation " (version in 2010) appendix.
2.4 diagnostic test: vaccine is diluted to the viral suspension that every ml contains the minimal infecting dose (MID) of 100 rabbits with normal saline, fully mixes, put 10-15 ℃ of effect 60 minutes with the swine fever virus resistant specific serum of equivalent, during jolting 2-3 time.Neutralization is inoculated 2 of rabbits respectively after finishing, and establishes the contrast of virus control and normal saline simultaneously.Every rabbit ear vein injection 1.0ml, temp measuring method and body temperature reaction normal are with the efficacy test item.
2.5 exogenous virus check: test for 40 pages according to " Chinese people republic veterinary drug allusion quotation " (version in 2010) appendix.
2.6 safety verification: 4 batches of Seedlings are diluted to 6 part/ml with normal saline with vaccine, 4 of the healthy susceptible pigs of every batch of vaccine difference intramuscular injection, every 5ml (containing 30 parts).After annotating Seedling, morning and afternoon every day, each thermometric was observed once, observed 21.
2.7 efficacy test: select method with test-free; Vaccine is diluted to variable concentrations with sterile saline; 2 of the rabbits of auricular vein injection body weight 1.5-3.0kg, every 1.0ml carries out according to " Chinese people republic veterinary drug allusion quotation " (version in 2010) 72 pages.
2.8 residual moisture is measured: test for 38 pages according to " Chinese people republic veterinary drug allusion quotation " (version in 2010) appendix.
2.9 vacuum is measured: test for 48 pages according to " Chinese people republic veterinary drug allusion quotation " (version in 2010) appendix.
3, result
3.1 physical behavior check: 4 batches of vaccines of preparation, it is the spongy loose agglomerate of milky, is prone to break away from the bottle wall, add diluent after dissolving rapidly, free from extraneous odour.
3.2 steriling test: test for 42 pages according to " Chinese people republic veterinary drug allusion quotation " (version in 2010) appendix, made 4 batches of vaccines are asepsis growth.
3.3 mycoplasma check: test for 49 pages according to " Chinese people republic veterinary drug allusion quotation " (version in 2010) appendix, made 4 batches of vaccines are no mycoplasma and grow.
3.4 diagnostic test: the result of 4 batches of vaccine samples, except that thermal response appearred in positive controls, thermal response did not all appear in all the other groups in the 120h after inoculation.
3.5 exogenous virus check: test for 40 pages according to " Chinese people republic veterinary drug allusion quotation " (version in 2010) appendix, made 4 batches of vaccines are no exogenous virus pollution.
3.5 safety verification: the result has no significant change before showing each batch test pig body temperature, spirit, appetite and notes Seedling, judges that vaccine is qualified.
3.6 efficacy test: select method with test-free; Vaccine is diluted to variable concentrations with sterile saline; 2 of the rabbits of auricular vein injection body weight 1.5-3.0kg, every 1.0ml carries out according to " Chinese people republic veterinary drug allusion quotation " (version in 2010) 72 pages.The result sees table 4, and vaccine potency is up to the standards, and the rabbit infective dose that every part of live vaccines of hog cholera of suspension culture preparation is contained is 20 times that traditional rolling bottle is cultivated.
Table 4 rabbit efficacy test result
Annotate: ++ be the typing thermal response ,+be that slight fever is reacted ,-be reactionless.
3.7 residual moisture is measured: test for 38 pages according to " Chinese people republic veterinary drug allusion quotation " (version in 2010) appendix, made 4 batches of vaccines are all up to specification.
3.8 vacuum is measured: test for 48 pages according to " Chinese people republic veterinary drug allusion quotation " (version in 2010) appendix, made 4 batches of vaccines are all up to specification.
4 brief summaries
According to the check of " Chinese people republic veterinary drug allusion quotation " (version in 2010) live vaccines of hog cholera (cell source) product inspection method; Comparative test result shows the live vaccines of hog cholera with the microcarrier suspension culture system preparation; Every part is contained 15000 rabbit infective doses; And every part of swine Fever Vaccine that traditional rolling bottle method makes is only contained 750 rabbit infective doses, so the live vaccines of hog cholera effectiveness aspect of the inventive method preparation is more outstanding.
Claims (10)
1. a method that adopts the oscillatory type bioreactor to prepare live vaccines of hog cholera is characterized in that comprising the following steps:
1) cultivates seedling and use cell
With seedling with cell inoculation in the bioreactor jar that contains cell culture fluid and microcarrier; Start cell absorption program, behind oscillatory type absorption 3～4h or the cell adsorption rate reach 80% when above, be converted into the cell culture program; Carry out the oscillatory type cell culture, treat that cell grows to 1.5 * 10
8～3 * 10
8Individual/as during the g microcarrier, to stop to cultivate;
2) breeding seedling venom
The swine fever virus suspension inoculation in the step 1) cultured cells, behind inversion absorption 0.5～1h, is carried out the oscillatory type Virus culture, and per 1～2d gathers in the crops viral liquid, and in time changes and keep liquid, and harvesting frequency is 15～20 times, gathers the viral liquid of results;
3) will add freeze drying protectant through the viral liquid clarification filtration that is up to the standards, fully quantitatively packing behind the mixing, lyophilizing promptly obtains live vaccines of hog cholera.
2. the method for claim 1 is characterized in that, said bioreactor is a BelloCell type oscillatory type cell microcarrier suspension culture bioreactor.
3. the method for claim 1 is characterized in that, BioNoc II microcarrier density is 11g/L in the step 1) bioreactor jar, and inserting cell concentration is 1 * 10
7/ g～1 * 10
8/ g microcarrier.
4. the method for claim 1 is characterized in that, step 1) oscillatory type cell absorption program parameter is: cell culture fluid is 1.0mm/s through the rate of climb of microcarrier, and the top stops 25s, and decrease speed is 1.0mm/s, and the bottom stops 0s; Oscillatory type cell culture program parameter is: cell culture fluid is 1.5mm/s through the rate of climb of microcarrier, and the top stops 0s, and decrease speed is 1.5mm/s, and the bottom stops 60s.
5. method according to claim 1 is characterized in that step 2) in inoculation 10%～20% hog cholera lapinised virus strain cell seed culture of viruses or 1%～2% hog cholera lapinised virus strain spleen seed culture of viruses, said percentage ratio is the volume ratio of seed culture of viruses and virus-culturing fluid.
6. the method for claim 1 is characterized in that step 2) oscillatory type Virus culture parameter is virus-culturing fluid through microcarrier the rate of climb is 1.0mm/s that the top stops 10s; Virus-culturing fluid is through the decrease speed 1.0mm/s of microcarrier, and the bottom stops 30s.
7. the method for claim 1 is characterized in that step 2) the every 1ml of viral liquid that obtains after the Virus culture contains virus >=1000000 a rabbit infective dose.
8. method according to claim 1 is characterized in that said seedling uses cell to be PK15 cell line; Cell culture fluid is the DMEM that contains 5%～8% hyclone, and virus-culturing fluid is the MEM that contains 1～2% hyclone, and pH value is 7.0～7.4; Cultivation temperature is 36.5～37 ℃, CO
2Concentration 1%～5%.
9. the method for claim 1 is characterized in that the freeze drying protectant described in the step 3) is 5% sucrose skim milk.
10. the live vaccines of hog cholera of the arbitrary described method preparation of claim 1-9.
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| CN201110412446.1 | 2011-12-12 | ||
| CN201210181238XA CN102743748A (en) | 2011-12-12 | 2012-06-04 | Method for preparing swine fever live vaccine by using oscillatory type bioreactor |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103656660A (en) * | 2013-11-08 | 2014-03-26 | 中牧实业股份有限公司 | Heat-resistant freeze-drying protective agent for animal live vaccine, preparation method and application thereof |
| CN107058211A (en) * | 2016-12-22 | 2017-08-18 | 武汉市工程科学技术研究院 | Carry ST cell lines and its application of rabbitization swine fever low virulent strain virus |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101926991A (en) * | 2010-01-28 | 2010-12-29 | 洛阳普莱柯生物工程有限公司 | Classical swine fever virus vaccine and production method thereof |
-
2012
- 2012-06-04 CN CN201210181238XA patent/CN102743748A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101926991A (en) * | 2010-01-28 | 2010-12-29 | 洛阳普莱柯生物工程有限公司 | Classical swine fever virus vaccine and production method thereof |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103656660A (en) * | 2013-11-08 | 2014-03-26 | 中牧实业股份有限公司 | Heat-resistant freeze-drying protective agent for animal live vaccine, preparation method and application thereof |
| CN103656660B (en) * | 2013-11-08 | 2017-04-05 | 中牧实业股份有限公司 | A kind of animal heat-resisting lyophilized protecting agent of live vaccine, its preparation method and application |
| CN107058211A (en) * | 2016-12-22 | 2017-08-18 | 武汉市工程科学技术研究院 | Carry ST cell lines and its application of rabbitization swine fever low virulent strain virus |
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Application publication date: 20121024 |