CN102988972A - Method for producing porcine parvovirus inactivated vaccine by using torrent bioreactor - Google Patents
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Abstract
The invention relates to a method for producing a porcine parvovirus inactivated vaccine by using a torrent bioreactor, comprising the following steps: inoculating the porcine parvovirus into the IBRS-2 cell, and culturing to obtain a production virus strain; inoculating the IBRS-2 cell into the torrent bioreactor; inoculating the prepared porcine parvovirus strain into the IBRS-2 cell cultured in the torrent bioreactor, and proliferating and culturing the virus; harvesting the proliferated virus solution; inactivating; and matching to obtain the porcine parvovirus inactivated vaccine. According to the method, because the torrent bioreactor and a paper vector are used, the shearing force generated by a microcarrier bioreactor used for culturing the cell is avoided, the density and the viability of the cell are further improved, and the capacity of proliferating the viruses by the cell is enhanced. The reactor can be used for monitoring and automatically regulating the optimal condition for growing the cells, thus the viruses harvested in all bathes have small difference, the quality stability of the vaccine is improved, the vaccine production efficiency is improved, and the vaccine production cost is reduced.
Description
Technical field
The present invention relates to belong to veterinary biologics and cultivate technical field, be specifically related to a kind of method of using the rip current type bioreactor to produce porcine parvovirus inactivated vaccines.
Background technology
Porcine parvovirus (porcine parvovirus, PPV) be cause the sow breeding difficulty as long as one of disease.Main manifestations is infected sow, particularly the negative multiparity of multiparity sow and serology is miscarried, produce stillborn fetus, monster, mummy tire, weak tire and Repeat breeding etc., PPV also has harm to fetus after infecting, and does not generally show obvious clinical symptoms after the pig infection to other ages.This disease worldwide extensively distributes, and it is popular that great majority infection pig farm is endemicity, is difficult to purify after swinery infects, thereby causes lasting economic loss, seriously hindered the sound development of global pig industry.PPV serotype is single, rare variation, but still do not have at present the medicine that can effectively treat, therefore, this sick immunoprophylaxis just seems even more important.In recent years, using vaccine is the unique method of prevention porcine parvovirus infection, raising sow breeding potential.
Tradition rolling bottle production technology is ripe at present IBRS-2 and other vaccine stroma cell culture processes.But because the method complex operation, the link that goes down to posterity is many, and product is easily contaminated, and the defectives such as the difficult control of product quality are for its large-scale application is brought obstacle.In recent years, also there is the scholar to use the microcarrier bioreactor to carry out cell and cultivation virus, and then produces vaccine.The widest with microcarrier Cytodex serial application, but microcarrier all has complicated treatment step before and after using, and the propagation of cell also is subject to the factor affecting such as shearing force, the accumulation of metabolite, is difficult to equally large-scale application.
Summary of the invention
Purpose of the present invention is exactly for the deficiencies in the prior art, and a kind of method of using the rip current type bioreactor to produce porcine parvovirus inactivated vaccines proposed, by the method can solve that production of vaccine efficient is low, unstable product quality, virus titer are low, complex operation, the link that goes down to posterity are many, the easy problem such as contaminated of product.
Technical scheme of the present invention realizes as follows:
(1) preparation of pig parvoviral kind poison: the IBRS-2 cell spinner bottle that will recover from liquid nitrogen goes down to posterity, when treating that cell grows up to monolayer, discard culture fluid, pig parvoviral is inoculated into the IBRS-2 cell, dosage of inoculation is 0.01MOI, changes the maintenance medium of 2% serum after the inoculation and cultivates, and treats that the IBRS-2 cytopathy reaches 80% and gathers in the crops virus liquid when above, the virus liquid of results is carried out virus titer mensuration and steriling test, the TCID of virus liquid
50〉=10
6.5TCID
50/ ml, aseptic detection is qualified in pig parvoviral kind poison.
(2) IBRS-2 cell inoculation rip current type bioreactor: the IBRS-2 cell of first tube being cultivated digests with trypsin, and when the Cell sap cumulative volume that digests is 4 liters, number of cells reaches about 2 * 10
9The time, Cell sap is squeezed in the scraps of paper carrier of rip current type reactor with peristaltic pump, carry out the breeding of cell and cultivate, the Temperature Setting of rip current type bioreactor is 37 ℃ during cultured cell, and pH is set as 7.2～7.4, and dissolved oxygen is set as 30 ~ 60%.
(3) bioreactor cell Pigs Inoculated parvovirus: cell density reaches 1 * 10 in the rip current type bioreactor
7During individual cell/ml, the pig parvoviral kind poison of preparation is inoculated into the IBRS-2 cell of cultivating in the rip current type bioreactor, dosage of inoculation is 0.01MOI, standing adsorption 1h after the inoculation, rip current type bioreactor design temperature is 36.5 ℃ during Virus culture, pH is set as 7.4～7.6, and dissolved oxygen is set as 30 ~ 60%, agitator rotating speed 55rpm/min.
(4) connect behind the poison behind the 60h, the virus liquid of results breeding, deactivation is joined Seedling and is namely obtained porcine parvovirus inactivated vaccines.
Wherein in the step of the present invention (3) before the bioreactor virus inoculation, should be first that culture fluid in the bioreactor is all emptying, with the aseptic PBS circulation flushing cell of preheating 2 times, emptying PBS, with peristaltic pump the kind of preheating is beaten cruelly in the infusion bag again, inoculate in the torrent bag of backward bioreactor and squeeze into culture medium.
Wherein the results of the virus liquid described in the step of the present invention (4) comprise cell conditioned medium liquid and contain virus liquid in the infusion bag of scraps of paper carrier, to contain the virus liquid centrifugal removal cell debris behind 3 multigelations in the infusion bag of scraps of paper carrier, mix with the supernatant of cell again, be positioned over-20 ℃ and save backup.The hybrid virus liquid of results is measured virus titer and red cell agglutination valency, and every milliliter of viral level is not less than 10 in the virus liquid
8.0TCID
50, virus liquid is not less than the available of 1:512 to the guinea-pig red blood cell hemagglutinative titer.
Wherein the deactivation described in the step of the present invention (4) is to use formalin-inactivated, in virus liquid, add 40% formalin, the limit edged mixes, when the formalin final concentration is 0.3% to the mixed liquor, stop to add, then inactivation of virus liquid is placed 37 ℃ of greenhouses to place 48h, during jolting 3～4 times, then inactivation of virus liquid is placed 2～8 ℃ of preservations.
Wherein the Seedling of joining described in the step of the present invention (4) is that the oil phase that contains white oil is even with the water emulsifying that contains virus liquid, step is for to squeeze into oil phase in the emulsion tank, start mixer to the 2000r/min stirring that slowly runs, slowly add simultaneously water, carry rotating speed after adding to 2900r/min emulsifying 30～100min, after the emulsifying, got about 10ml with 3000r/min centrifugal 15 minutes, if lamination is arranged, should repeat emulsifying once.
Above-described oil phase is with injection white oil 94 weight portions, and aluminium stearate 0.5 weight portion mixes, and then adds 6 weight portion span-80, makes at 121 ℃ of lower autoclaving 30min.Described water is virus liquid 96 weight portions that preservation is no more than 1 month, and it is even that the ofloxacin of the sodium azide of 0.01 weight portion and 0.015 weight portion mixes rolling, then adds the tween 80 of 20～30 ℃ of 4 weight portions, and shake well is made to dissolving fully.
The beneficial effect that technical solutions according to the invention are brought is:
1, the rip current type bioreactor adopts scraps of paper carrier training mode, and scraps of paper carrier is 3-D solid structure, can increase cell Absorption Growth area.This pattern is easy to Growth of Cells and nutrition supply, incubation can adopt continuous perfusion culture, discharge old liquid when constantly adding fresh medium, can supply with timely and effectively nutrition and remove metabolite, make cell be in all the time good nutritional status, be applicable to high-density cells and cultivate, prolong simultaneously cell and hold time, be conducive to the lasting breeding of virus, improve vaccine output.
2, compare with torrent filling type bioreactor scraps of paper carrier system with traditional rolling bottle cell culture pig parvoviral and have following advantage: can solve that production efficiency is low, unstable product quality, problem that virus titer is low, by the change of production technology and production technology, but the Quality and yield of General Promotion vaccine; Can reduce production costs in a large number, and can shorten the production cycle; Use the torrent filling type bioreactor and have the automaticity height, total enclosing, the production procedure of channelization system and process automation monitoring, control technology, not only reduced the chance of contamination of cells, and in the force intensity that lightens one's labor, reduce the manual operation factor affecting, the production technology simple and stable is easy to operate, the large occupied ground of output is little, be easy to expand the scale of production fast, quality is easy to realize the advantages such as equalization stable; The applying biological reactor assembly, the virus titer of product improves more than 10 times than traditional roller system, and 3 ~ 4 times of costs.
The specific embodiment
In order to make technical scheme of the present invention clearer, the present invention is further illustrated below in conjunction with specific embodiment.
Embodiment instrument and material:
Bioreactor: the Hangzhou An Pu AP20C of company model, the maximum functional volume is 10L.
Carrier: scraps of paper carrier, every bag contains scraps of paper carrier 150g, available from Hangzhou An Pu company.
Pig parvoviral: CP-99 strain.
Cell growth medium: containing volumetric concentration is the DMEM(Beijing Qingdatianyi Bioisystech Co., Ltd of 10% new-born calf serum).
The virus maintenance medium: containing volumetric concentration is the DMEM(Beijing Qingdatianyi Bioisystech Co., Ltd of 2% new-born calf serum).
Embodiment 1: rip current type bioreactor culture IBRS-2 cell
1, cell recovery
From liquid nitrogen container, take out the IBRS-2 cell cryopreservation tube, put into rapidly 37 ℃ of water, constantly rock it is thawed rapidly, separate out cell suspension with pipettor, pack in the aseptic centrifuge tube, add 9ml cell growth medium (serum-free), it is centrifugal to put into centrifuge, centrifugal 10 minutes of 1000rpm, abandon supernatant, add cell culture fluid (containing 10% new-born calf serum) cell is hanged, move in the disposable Tissue Culture Flask, put 37 ℃ 5% CO
2Cultivate in the constant incubator, change cell growth medium once after 6 hours, continue to cultivate.
2, passage
Get well-grown IBRS-2 cell monolayer, discard culture medium, use the PBS washed twice, add an amount of tryptic digestive juice, keep flat about 2min, observation of cell digestion situation, if it is smudgy to find that cellular layer occurs, then discard pancreatin, add an amount of culture fluid (DMEM that contains 10% serum), blow and beat gently to cell with glass pipette and be separated into individual cells.Seed cell bottle number be vaccinated cell bottle number and can go down to posterity in the ratio of 1:3.IBRS-2 cell after going down to posterity is put 37 ℃ 5%CO
2Constant incubator in cultivate.
3, cultivation and the amplification of the kind subchain of IBRS-2 cell
When cell covers with 90% when above, use trypsin digestion cell, be seeded in the 20 ml scraps of paper tube reactors (containing scraps of paper carrier 0.6g) with 20 ~ 30ml growth-promoting media re-suspended cell, inoculum concentration is 5.0 * 10
6Individual tube incubator rotating speed is 35r/min, change the culture medium in the tube reactor every day, and the variation of glucose content in the detection incubation, when sugared consumption tends to be steady, discard culture medium, add 20ml PBS submergence rinse 2 times, add the 15ml pancreatin, 37 ℃ of lower rotation 20min that continue, thermal agitation after taking out, collecting cell is put into the receiving flask that adds culture medium, counts with cell counting count board.
The cell of gathering in the crops in 1 20 ml scraps of paper tube reactor is forwarded in 1 220 ml scraps of paper tube reactor (containing scraps of paper carrier 5g), and inoculum concentration is about 5.0 * 10
7Individual, the culture fluid volume is 200 ~ 300ml, and condition of culture is identical with 20ml scraps of paper tube reactor with digestion method.It is that 4L contains in the culture bag of scraps of paper carrier (containing scraps of paper carrier 150g) that the cell that digests in 3 220ml tubes is inoculated into volume with peristaltic pump, and inoculum concentration is about 2 * 10
9Individual, standing adsorption 1h after the inoculation, after will contain scraps of paper carrier culture bag be the perfusion tank of the torrent filling type bioreactor of the infusion bag maximum volume 10L that puts into the general AP20C model of peace, the control condition of culture is 37 ℃ of temperature, PH 7.2 ~ 7.4, dissolved oxygen DO 30% ~ 60%, agitator rotating speed 55rpm/min, 250mL/min begins the 30min that circulates.Get 2 sample samples behind the 350mL/min circulation 30min, a microscopy that is used for, a being used for surveyed sugar, as initial sugared concentration.In visual field of microscopy, be less than 10 cell number and then begin the 450mL/min normal circulation.
The torrent filling type bioreactor has automatic control to cultivate function.Can determine flow feeding and change the liquid situation that glucose is maintained more than the 2g/L, and cell counting is carried out in regularly sampling, when cell density reaches 1 * 10 by monitoring residual sugar amount
7During individual cell/ml, can connect poison.Cell culture needs 5 day time altogether, a few days ago the time, surveys sugar every sampling in 24 hours.Cultivate rear 3 days, sugar was surveyed in sampling in per 12 hours, needed entirely change at the 3rd day that cultivates liquid according to the sugared situation of consumption.
Described scraps of paper carrier is polyesteramide scraps of paper carriers, and scraps of paper carrier needs simple pretreatment before use, and method is as follows: soak scraps of paper carrier with aseptic PBS and spend the night; With aseptic PBS wash cycles scraps of paper carrier 3 times; Soaking scraps of paper carrier with the serum-free cell culture growth-promoting media spends the night and gets final product.
Embodiment 2: rip current type bioreactor culture pig parvoviral cell
1, the preparation of pig parvoviral kind poison
First the IBRS-2 cell spinner bottle is gone down to posterity, when treating that cell grows up to monolayer, discard culture fluid, Pigs Inoculated parvovirus CP-99 strain in the cell, dosage of inoculation is 0.01MOI, the maintenance medium of changing 2% serum after the inoculation continues to cultivate.Treat that cytopathy reaches 80% above time results virus liquid, the virus liquid of results is carried out virus titer measure and steriling test the TCID of virus liquid
50〉=10
6.5TCID
50/ ml, qualified can be used as of aseptic detection produced kind of a poison.
2, bioreactor cell Pigs Inoculated parvovirus
When rip current type bioreactor cell continuous culture 5 days, to survey consumption sugar amount and reach when stablizing, cell density generally reaches 1 * 10
7Carry out the inoculation of virus during the individual cell/ml left and right sides.First that culture fluid is all emptying during virus inoculation, the aseptic PBS circulation flushing cell of usefulness preheating 2 times, emptying PBS beats the kind of preheating in the infusion bag cruelly with peristaltic pump again, and connecing the toxic agent amount is 0.01MOI, and volume is 4 liters.Standing adsorption 1h after the inoculation.At this moment, 2 liters of the culture medium of 2% aseptic new-born calf serum are squeezed in the torrent bag, adjusted parameter, 36.5 ℃ of temperature, pH 7.4 ~ 7.6, dissolved oxygen DO 30% ~ 60%, agitator rotating speed 55rpm/min.Behind the viruses adsorption 1h, with the normal speed circulation of 450mL/min.
3, results pig parvoviral
Sugar is surveyed in sampling in per 12 hours after connecing poison on the reactor, and glycosyl originally exhausts during 24h, namely adds concentrated solution and glucose since 24 hours needs, about fluid infusion cumulative volume 0.5L every day, glucose content is reached about 4g/L.Connect poison beginning in rear 48 hours sampling in per 6 hours, survey consumption sugar amount descends and tends towards stability.By the time when meeting the rear 60h of poison, toxic amount is the highest in the cell conditioned medium liquid, at this moment, the results virus liquid.
Step is: toxic maintenance medium in the torrent bag is got in the sterile chamber by peristaltic pump.4 liters of virus liquids of infusion bag are discharged 2 liters, remain 2 liters of virus liquids and stay in the bag, will contain in the infusion bag of virus liquid and put into-20 ℃ of refrigerator-freezer multigelations 3 times, be discharged in the sterile chamber virus liquid is aseptic again.Sampling after viral supernatant and cytopathy venom mixed detects malicious valency and red cell agglutination valency, and every milliliter of viral level is not less than 10
8.0TCID
50, virus liquid is not less than 1:512 to the guinea-pig red blood cell hemagglutinative titer.
Embodiment 3: the optimization Test of pacifying PPV proliferation conditions in the general AP20C rip current type bioreactor
1, cell inoculation quantity is on the impact of PPV propagation
3 groups of (A, B, C group) different cell inoculum concentrations are selected in test, are respectively 1 * 10
6Cells/ml, 1 * 10
7Cells/ml, 2 * 10
7Cells/ml, 54h, 60h, 66h, 72h time results virus liquid and measure malicious valency behind virus inoculation, research cell inoculation quantity is on the impact of PPV propagation.
Experimental data:
The different cell densities of table 1 are on the impact of PPV propagation
Experimental result shows that the malicious valency of results virus liquid is higher behind the virus inoculation 60h, and cell density is 1 * 10
7In the time of behind the cells/ml, virus liquid poison valency is higher.
2, connect the toxic agent amount to the impact of PPV proliferation conditions
Stablize when the cell sugar consumption rate and to connect the poison operation when constant, the different toxic agent amounts that connect of research are on the impact of PPV poison valency, and test is divided into three groups, meet poison amount MOI and be respectively 0.001,0.01,0.05, respectively at after the inoculation 48,54,60,66,72h sampling, measure malicious valency, the PPV that connects of an institute poison valency is 10
6.5TCID
50/ ml.Experimental result sees Table 2.
Receiving malicious method is: test is divided into 3 groups (A, B, C groups), and A surveys the malicious valency of the cell conditioned medium liquid of collecting; The B group is surveyed the malicious valency after the maintenance medium of staying 2 liters in the infusion bag is carried out 3 freeze thawing; The C group is surveyed the malicious valency of A, B group biased sample.Experimental data sees Table 3.
Table 2 difference connects the different receipts poison times of toxic agent amount to the impact of PPV propagation
Experimental result shows that the malicious valency of results virus liquid is higher behind the virus inoculation 60h, and connecing poison amount (MOI) is 0.01 o'clock, and virus liquid poison valency is higher.
Viral level in table 3 different solutions
Experimental result shows that infusion bag and both mixed liquor poisoning valencys are all higher after cell conditioned medium, the freeze thawing.
Embodiment 4: the preparation of porcine parvovirus inactivated vaccines
1, the deactivation of the pig parvoviral liquid of results
Virus liquid hybrid filtering among the embodiment 2 that is up to the standards in sterile chamber, is added 40% formalin, and the limit edged mixes, make that formalin concentration is 0.3% in the mixed liquor, then put 37 ℃ of effect 48h, during jolting 3～4 times, inactivation of virus liquid is put 2～8 ℃ of preservations, should be no more than 1 month.
2, oil emulsion vaccine preparation
(1) oil phase preparation: get injection white oil 94 weight portions, add aluminium stearate 0.5 weight portion, after the mixing, add 6 weight portion span-80,121 ℃ of 30min autoclavings are for subsequent use.
(2) water preparation: get virus liquid 96 weight portions, the ofloxacin rolling that adds 0.01% sodium azide and 0.015% is even, then adds tween 80 4 weight portions about 20～30 ℃, shake well, tween 80 is dissolved fully till.
(3) emulsifying: oil phase is squeezed in the emulsion tank, started mixer to the 2000 r/min(35.5Hz) stirring that slowly runs adds water simultaneously slowly, carries rotating speed to 2900r/min(47.5Hz after adding) emulsifying 30～100min.After the emulsifying, got about 10ml with 3000r/min centrifugal 15 minutes, if lamination is arranged, should repeat emulsifying once.If without layering, i.e. porcine parvovirus inactivated vaccines is finished in preparation.
Embodiment 5: pacify the contrast of general AP20C rip current type bioreactor culture IBRS-2 cells produce pig parvoviral and spinner culture IBRS-2 cells produce pig parvoviral.
1, spinner culture IBRS-2 cells produce pig parvoviral.
The IBRS-2 cell of recovery is used in the 75T Tissue Culture Flask and contains 10% new-born calf serum DMEM culture medium culturing, continuous passage is expanded to the 10L rolling bottle, the ratio that goes down to posterity 1:3, every 2d goes down to posterity 1 time, cultured cell becomes good monolayer to change viral maintenance medium, dose inoculation pig parvoviral by 0.01MOI carries out maintain, keeps situation according to cytopathy and gathers in the crops virus liquid.
2, the general AP20C rip current type bioreactor culture IBRS-2 cells produce pig parvoviral of peace
Prepare pig parvoviral liquid by the method in embodiment 1 and 2.
3, cell culture result contrast
Spinner culture technique cultured cell density when connecing poison is 1.0 * 10
5/ ml, volume are 1L, and cell density is 1 * 10 when using the general AP20C rip current type bioreactor of peace to connect poison
7Cells/ml, volume are 4L.Number of computations compares, and the cell quantity of 1 general AP20C bioreactor culture of peace is equivalent to 400 rolling bottles.
4, Virus culture Contrast on effect
The virus liquid poison valency of rolling bottle explained hereafter is 10
6.5TCID
50/ ml, hemagglutinative titer are 1:128.Use the average malicious valency of the general AP20C rip current type bioreactor virus liquid of peace 〉=10
8.0TCID
50/ ml, hemagglutinative titer are 1:512.Comparing result shows that the cell venom of the more traditional spinner culture production of virus liquid titre of using the general AP20C rip current type bioreactor propagation of peace is increased to few 1 more than the titre.
Claims (7)
1. method of using the rip current type bioreactor to produce porcine parvovirus inactivated vaccines is characterized in that production method and step are as follows:
(1) preparation of pig parvoviral kind poison: the IBRS-2 cell spinner bottle that will recover from liquid nitrogen goes down to posterity, when treating that cell grows up to monolayer, discard culture fluid, pig parvoviral is inoculated into the IBRS-2 cell, dosage of inoculation is 0.01MOI, changes the maintenance medium of 2% serum after the inoculation and cultivates, and treats that the IBRS-2 cytopathy reaches 80% and gathers in the crops virus liquid when above, the virus liquid of results is carried out virus titer mensuration and steriling test, the TCID of virus liquid
50〉=10
6.5TCID
50/ ml, aseptic detection is qualified in pig parvoviral kind poison;
(2) IBRS-2 cell inoculation rip current type bioreactor: the IBRS-2 cell of first tube being cultivated digests with trypsin, and when the Cell sap cumulative volume that digests is 4 liters, number of cells reaches about 2 * 10
9The time, Cell sap is squeezed in the scraps of paper carrier of rip current type reactor with peristaltic pump, carry out the breeding of cell and cultivate, the Temperature Setting of rip current type bioreactor is 37 ℃ during cultured cell, and pH is set as 7.2～7.4, and dissolved oxygen is set as 30 ~ 60%;
(3) bioreactor cell Pigs Inoculated parvovirus: cell density reaches 1 * 10 in the rip current type bioreactor
7During individual cell/ml, the pig parvoviral kind poison of preparation is inoculated into the IBRS-2 cell of cultivating in the rip current type bioreactor, dosage of inoculation is 0.01MOI, standing adsorption 1h after the inoculation, rip current type bioreactor design temperature is 36.5 ℃ during Virus culture, pH is set as 7.4～7.6, and dissolved oxygen is set as 30 ~ 60%, agitator rotating speed 55rpm/min;
(4) meet malicious 60h after, the virus liquid of results breedings, deactivation is joined Seedling and is namely obtained porcine parvovirus inactivated vaccines.
2. require 1 described a kind of method of using the rip current type bioreactor to produce porcine parvovirus inactivated vaccines according to patent, it is characterized in that: in the step (3) before the bioreactor virus inoculation, should be first that culture fluid in the bioreactor is all emptying, with the aseptic PBS circulation flushing cell of preheating 2 times, emptying PBS, with peristaltic pump the kind of preheating is beaten cruelly in the infusion bag again, inoculate in the torrent bag of backward bioreactor and squeeze into culture medium.
3. require 1 described a kind of method of using the rip current type bioreactor to produce porcine parvovirus inactivated vaccines according to patent, it is characterized in that: the results of the virus liquid described in the step (4) comprise cell conditioned medium liquid and contain virus liquid in the infusion bag of scraps of paper carrier, and every milliliter of viral level is not less than 10 in the virus liquid
8.0TCID
50, virus liquid is not less than the available of 1:512 to the guinea-pig red blood cell hemagglutinative titer.
4. require 1 described a kind of method of using the rip current type bioreactor to produce porcine parvovirus inactivated vaccines according to patent, it is characterized in that: the deactivation described in the step (4) is to use formalin-inactivated, in virus liquid, add 40% formalin, the limit edged mixes, when the formalin final concentration is 0.3% to the mixed liquor, stop to add, then inactivation of virus liquid is placed 37 ℃ of greenhouses to place 48h, then during this time jolting 3～4 times places inactivation of virus liquid 2～8 ℃ of preservations.
5. require 1 described a kind of method of using the rip current type bioreactor to produce porcine parvovirus inactivated vaccines according to patent, it is characterized in that: the Seedling of joining described in the step (4) is that the oil phase that contains white oil is even with the water emulsifying that contains virus liquid, step is for to squeeze into oil phase in the emulsion tank, start mixer to the 2000r/min stirring that slowly runs, slowly add simultaneously water, carry rotating speed after adding to 2900r/min emulsifying 30～100min, after the emulsifying, got about 10ml with 3000r/min centrifugal 15 minutes, if lamination is arranged, should repeat emulsifying once.
6. require 5 described a kind of methods of using the rip current type bioreactor to produce porcine parvovirus inactivated vaccines according to patent, it is characterized in that: described oil phase is with injection white oil 94 weight portions, aluminium stearate 0.5 weight portion mixes, and then add the span-80 of 6 weight portions, make at 121 ℃ of lower autoclaving 30min.
7. require 5 described a kind of methods of using the rip current type bioreactor to produce porcine parvovirus inactivated vaccines according to patent, it is characterized in that: described water is virus liquid 96 weight portions that preservation is no more than 1 month, 0.01 it is even that the ofloxacin of the sodium azide of weight portion and 0.015 weight portion mixes rolling, then the tween 80 that adds 20～30 ℃ of 4 weight portions, shake well is made to dissolving fully.
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| CN103877572A (en) * | 2014-03-21 | 2014-06-25 | 吉林正业生物制品股份有限公司 | Method for preparing porcine parvovirus disease inactivated vaccine |
| CN104498446A (en) * | 2014-12-15 | 2015-04-08 | 北京科兴生物制品有限公司 | Method for inactivating virus in vaccine production |
| CN105039264A (en) * | 2015-07-14 | 2015-11-11 | 天津瑞普生物技术股份有限公司 | Canine parvovirus proliferation method |
| CN107802830A (en) * | 2017-10-19 | 2018-03-16 | 吉林农业科技学院 | A kind of pig parvoviral disease vaccine emulsification method based on mineral oil adjuvant |
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| CN104498446A (en) * | 2014-12-15 | 2015-04-08 | 北京科兴生物制品有限公司 | Method for inactivating virus in vaccine production |
| CN104498446B (en) * | 2014-12-15 | 2017-03-15 | 北京科兴生物制品有限公司 | A kind of method of inactivation of viruses in production of vaccine |
| CN105039264A (en) * | 2015-07-14 | 2015-11-11 | 天津瑞普生物技术股份有限公司 | Canine parvovirus proliferation method |
| CN107802830A (en) * | 2017-10-19 | 2018-03-16 | 吉林农业科技学院 | A kind of pig parvoviral disease vaccine emulsification method based on mineral oil adjuvant |
| CN108310374A (en) * | 2017-12-30 | 2018-07-24 | 华南农业大学 | A kind of preparation method of avian influenza virus inactivated vaccine |
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Inventor after: Chen Shenmiao Inventor after: Niu Chengming Inventor after: Guo Xianpo Inventor after: Dong Xinrong Inventor after: Yang Lingzhi Inventor before: Xu Mingming Inventor before: Chen Shenmiao Inventor before: Dong Xinrong |
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