CN104367996A - Method for producing swine pseudorabies live vaccine by using passage cell source, and product thereof - Google Patents
Method for producing swine pseudorabies live vaccine by using passage cell source, and product thereof Download PDFInfo
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Abstract
The present invention provides a method for producing a swine pseudorabies live vaccine by using a passage cell source, and a swine pseudorabies live vaccine product thereof. The method comprises: adopting passage cells to culture a swine pseudorabies virus attenuated strain; harvesting to obtain a cell culture virus solution; and adding a stabilizer, and carrying out freezing vacuum drying to obtain the passage cell derived swine pseudorabies live vaccine. During the use, the diluent of the prepared vaccine of the present invention can further be used. According to the present invention, the method has advantages of simple and stable production process, easy operation, high virus content, low difference between batches, controllable quality, significant vaccine yield and quality improving, and the like, the obtained product can be stored for a long time at the room temperature, especially at the temperature of 2-8 DEG C, the shelf life can be 36 months, and the diluted vaccine obtained by diluting the vaccine with the diluent of the present invention can be stored for a long time at the room temperature and can maintain the stability.
Description
Technical field
The invention belongs to veterinary biologics technical field, more specifically relate to method and goods thereof that a kind of subculture cell source produces pseudorabies disease live-vaccine.
Background technology
Pseudorabies is a kind of acute infectious disease being caused many animals by pseudorabies virus (PRV).Main pig-raising countries on this infectious disease throughout world.Immunity inoculation is the main policies of control porcine pseudorabies.It is chick embryo fibroblast primary cell that China produces pseudorabies disease live-vaccine cell used at present, cause differences between batches comparatively large, it is not high to produce malicious titre, brings difficulty to the raising of vaccine output, effect, and cell debris and foreign protein many, easily cause allergic reaction.And depositing of semi-finished product virus liquid needs to preserve below-15 DEG C, in order to avoid cause the reduction of virus titer.
The protective agent that current live vaccine is conventional is gelatin, sucrose, skim milk, tryptone, EDTA, dipotassium hydrogen phosphate, potassium dihydrogen phosphate etc., complicated components, the more difficult collocation of each component ratio, so that protective value is poor.If under 2 DEG C ~ 8 DEG C conditions, storage life only has 3 ~ 6 months, need to preserve below-15 DEG C more.And the protectant development of domestic traditional animal vaccines relatively lags behind, the long-term preservation of vaccine and long-distance transport are very limited, add that epidemic prevention department of basic unit lacks necessary freezing and cold storage establishment, easily cause pestilence because of immuning failure.Current, China's pseudorabies live vaccine storage temperature remains less than-15 DEG C, so the research of heat resisting protective is badly in need of very much.
When existing freeze-dried live vaccine dilutes with diluent in use, be generally carry out at normal temperatures, need to carry out inoculation in the short period, avoid virus titer to decline, and then cause immuning failure.Existing pseudorabies disease live-vaccine normally dilutes with normal saline, carries out immunity after dilution in 30 minutes, 10 minutes even shorter time.
Summary of the invention
In order to overcome above-mentioned technological deficiency, the object of this invention is to provide method and pseudorabies disease live-vaccine product that a kind of subculture cell source produces pseudorabies disease live-vaccine.
For achieving the above object, the present invention provide firstly a kind of method that subculture cell source produces pseudorabies disease live-vaccine.The method comprises the steps:
(1) porcine pseudorabies virus low virulent strain is cultivated with passage cell;
(2) virus liquid is gathered in the crops;
(3) add stabilizing agent, obtain subculture cell source pseudorabies disease live-vaccine through lyophilisation.
Preferably, described step (1) comprises the steps:
(1a) the going down to posterity and cultivation of seedling cell: described passage cell, through pancreatin cell dispersal had digestive transfer culture, continues to cultivate with cell growth medium, forms passage cell monolayer;
(1b) breeding of cell seed culture of viruses: described porcine pseudorabies virus low virulent strain is seeded to the passage cell monolayer obtained in described step (1a), continues to cultivate with cell maintenance medium; After 24h ~ 44h, harvesting cultivates virus liquid as production seed culture of viruses;
(1c) breeding of seedling venom: get the passage cell monolayer obtained in described step (1a), discard described cell growth medium, inoculates the production seed culture of viruses obtained in described step (1b), continues to cultivate.
Preferably, the temperature used of the cell culture in described step (1a), (1b), (1c) is 36 DEG C ~ 38 DEG C.
Preferably, the inoculum concentration in described step (1b), (1c) during the inoculation of porcine pseudorabies virus low virulent strain is the maintenance medium of the Cells for production seed culture of viruses of 1% ~ 2% percent by volume.
Preferably, the cell growth medium in described step (1a) is containing the cell culture fluid of 90% ~ 97% percent by volume and the Ox blood serum of 3% ~ 10% volume ratio, and the pH value of described cell growth medium is 7.0 ~ 8.0.
Preferably, the cell maintenance medium in described step (1b), (1c) is containing the cell culture fluid of 95% ~ 99% percent by volume and the Ox blood serum of 1% ~ 5% volume ratio, and the pH value of described cell maintenance medium is 7.1 ~ 7.5.
The cell culture fluid that the passage cell of PRV (Pseudorabies virus) is easily bred in wherein said applicable cultivation include but not limited to be selected from MEM culture fluid, DMEM culture fluid, EMEM culture fluid, 199 culture fluid, RPMI-1640 and α-MEM culture fluid any one, described Ox blood serum includes but not limited to hyclone, new-born calf serum or calf serum.
Preferably, described cell culture fluid is MEM culture fluid, and described Ox blood serum is hyclone.
Preferably, after described step (2) comprises the steps: to connect poison, 24h ~ 44h harvesting cultivates venom, and the venom of results is placed in more than 0 DEG C preservation.
Preferred, preserve under the venom of described results is placed in 2 DEG C ~ 8 DEG C conditions.
The passage cell being suitable for porcine pseudorabies virus includes but not limited to, (ATCC numbers ST cell line: CRL-1746), (ATCC numbers PK-15 cell line: CCL-33), (ATCC numbers African green monkey kidney cell Marc-145 cell line: CRL-12219), (ATCC numbers bovine kidney cells MDBK cell line: CCL-22), (ATCC numbers bull testis passage cell BT cell line: CRL-1390), (ATCC numbers Vero cell line: CCL-81), (ATCC numbers BHK-21 cells: CCL-10), porcine kidney cell system (as: IBRS-2, see such as, DECASTRO, M.P.1964.Behavior offoot and mouth disease virus in cell culture:susceptibility of the IB-RS-2swine cell line.Arquivos Instituto Biologica31:63-78), rabbit kidney continuous cell line (RK, CCL-106) as: ATCC numbers: the continuous cell line such as.
Preferably, described passage cell is pig testis subculture cells ST, porcine kidney cell PK15 or porcine kidney cell IBRS-2.
Described porcine pseudorabies virus low virulent strain to be selected from porcine pseudorabies virus (Bartha K-61 strain), porcine pseudorabies virus (Bucharest strain), porcine pseudorabies virus (HB-98 strain), porcine pseudorabies virus gene-deleted strain (SA215 strain) one or more.
Preferably, described porcine pseudorabies virus low virulent strain is selected from porcine pseudorabies virus gene-deleted strain SA215 strain.
Described porcine pseudorabies virus gene-deleted strain SA215 strain, be preserved in China typical culture collection center, preservation date is on March 28th, 2000, and preservation address is Wuhan, China Wuhan University, deposit number is CCTCC No:V200002, is disclosed in patent CN101186902A.
The present invention's use " low virulent strain " one word, also claims " attenuated strain ".Usually, attenuation is realized by UV radiation, chemical treatment or external continuous high-order successive transfer culture; The change of outer aobvious gene, as made the specific nucleotide in known array lack or insert in viral genome by nucleotide, also may cause attenuation.Make virus lose pathogenic but keep antigenicity by these methods, the gene relating to pathogen analytic metabolism is suddenlyd change and manually reduces pathogen toxicity and the Strain that obtains.
The present invention's " live vaccine " used one word, refers to from attenuation but still the vaccine prepared of the virus of the cell of reproducible host organisms.
Two of object of the present invention is to provide a kind of pseudorabies disease live-vaccine goods.
These pseudorabies disease live-vaccine goods are prepared by said method, and these goods comprise stabilizing agent.
Preferably, described stabilizing agent comprises trehalose, L-sodium and vitamin C.
Preferably, the ratio that described trehalose accounts for stabilizing agent is 4% ~ 6%W/V, and the ratio that described L-sodium accounts for stabilizing agent is 4% ~ 6%W/V, and the ratio that described vitamin C accounts for stabilizing agent is 0.1% ~ 0.3%W/V.
Preferably, described stabilizing agent comprises polyvinylpyrrolidone further.
Preferably, the ratio that described polyvinylpyrrolidone accounts for stabilizing agent is 4% ~ 6%W/V.
Three of object of the present invention is to provide the application of a kind of pseudorabies disease live-vaccine in the medicine infected for the preparation of prevention and therapy porcine pseudorabies virus.
Pseudorabies disease live-vaccine goods prepared by preparation method of the present invention, comprise diluent further, during use, dilute pseudorabies disease live-vaccine with diluent.
Preferably, described diluent comprises sorbitol and sodium thiosulfate.
Preferably, the ratio that described sorbitol accounts for diluent is 1% ~ 3%W/V, and the ratio that described sodium thiosulfate accounts for diluent is 1% ~ 5%W/V.
Preferred, the ratio that described sorbitol accounts for diluent is 1.5%W/V, and the ratio that described sodium thiosulfate accounts for diluent is 3.5%W/V.
The present invention's predicate used " prevention " refers to by giving suppress Pseudorabies virus to infect according to vaccine combination of the present invention or postpone all behaviors of pseudorabies outbreak.Term " treatment " refers to all behaviors making Pseudorabies virus infect the symptom caused to alleviate or take a turn for the better by giving vaccine combination according to the present invention.
Compared with prior art, the method that subculture cell source of the present invention produces pseudorabies disease live-vaccine has that production technology is simple and stable, viral level is high, differences between batches are little, easy to control the quality, vaccine seed output and quality can be significantly improved, reduce the advantages such as anaphylaxis.The pseudorabies disease live-vaccine safety utilizing production method of the present invention to obtain is good, immune efficacy is high, have good immanoprotection action to porcine pseudorabies strong virus attack; The stabilizing agent raw material components that the present invention adopts is few, and cost is low, simple to operate, can large-scale production, and the lyophilizing pseudorabies live vaccine prepared can be preserved 36 months under 2 DEG C ~ 8 DEG C conditions, and virus titer decline is no more than 1 titre.During use, dilute lyophilizing pseudorabies live vaccine with diluent of the present invention, the longer time can be preserved at normal temperatures, still can keep its stability.
Detailed description of the invention
For making the present invention easier to understand, below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention, NM concrete test method in the following example, conveniently test method is carried out usually.
Term " head part " in the present invention refers to the amount of vaccine of every pig injection.
" TCID described in the present invention
50" (50%tissue culture infective dose) refer to half cell culture infective amount, is a kind of representation representing virus infectivity.
MEM fluid medium (liquid) the MEM dehydrated medium of purchased from American Life Technologies company is prepared according to its description.
Hyclone is purchased from PAA company.
The preparation of embodiment 1 heat resisting protective
The composition of heat resisting protective and constituent content are in table 1.
The composition content of table 1 heat resisting protective
For often kind of heat resisting protective, under agitation 1L water for injection is heated to the temperature of 60 DEG C, compound is added in hot water lentamente, to promote that it dissolves under maintenance is via the stirring of magnetism stick subsequently simultaneously.Maintain after adding the last time and stir about 20 minutes, thus cause the solution obtaining homogenizing.
Then make solution be cooled to room temperature, after cooling, heat resisting protective is by carrying out filtration sterilization, room temperature preservation via the aperture filter membrane with 0.22 μm.
Embodiment 2 passage cell produces the production technology of pseudorabies disease live-vaccine
Produce the production technology of pseudorabies disease live-vaccine with passage cell, concrete steps are as follows:
(1) select passage cell as seedling cell.
(2) the going down to posterity and cultivation of seedling cell: above-mentioned passage cell is through pancreatin cell dispersal liquid had digestive transfer culture, continue to cultivate under 36 DEG C ~ 38 DEG C conditions with the cell growth medium (pH is adjusted to 7.0 ~ 8.0) containing the MEM culture fluid of 90% ~ 97% percent by volume and the hyclone of 3% ~ 10% volume ratio, forming good monolayer, going down to posterity or virus inoculation for continuing.
(3) breeding of cell seed culture of viruses: by the well-grown above-mentioned passage cell monolayer of porcine pseudorabies virus low virulent strain inoculation, continue to cultivate in 36 DEG C ~ 38 DEG C with the cell maintenance medium (pH is adjusted to 7.1 ~ 7.5) containing the MEM culture fluid of 95% ~ 99% percent by volume and the hyclone of 1% ~ 5% volume ratio; After 24h ~ 44h, harvesting cultivates virus liquid as production seed culture of viruses.
(4) breeding of seedling venom: get the above-mentioned passage cell forming good monolayer, discard cell growth medium, inoculates above-mentioned toxic maintenance medium, continues to cultivate; After connecing poison, 24h ~ 44h harvesting cultivates venom, and the venom of results is placed in more than 0 DEG C preservation.
(5) join Seedling and lyophilizing: by the virus-culturing fluid of above-mentioned preparation, add stabilizing agent, fully shake up, get product after carrying out lyophilisation.
The preparation of embodiment 3 virus liquid
Select pig testis (ST) passage cell, Ren sus domestica (PK15) passage cell and Ren sus domestica (IBRS-2) passage cell, porcine pseudorabies virus low virulent strain is selected from porcine pseudorabies virus SA215 strain, porcine pseudorabies virus liquid B1, B2 and B3 is prepared respectively according to the production technology of embodiment 2, tire after measured, every 1ml viral level is respectively 10
9.8tCID
50, 10
9.75tCID
50, 10
9.8tCID
50, be placed in more than 0 DEG C preservation.
The storage life test of embodiment 4 virus liquid
Virus liquid B1, B2 and B3 of embodiment 3 being prepared are divided into 8 parts respectively, preserve, measure its storage life under being placed in-20 DEG C ,-15 DEG C, 0 DEG C, 2 DEG C, 4 DEG C, 8 DEG C, 25 DEG C and 37 DEG C of conditions.
The storage life of virus liquid under table 2 condition of different temperatures
As can be seen from Table 2, (in production, virus liquid was produced and was accomplished to the virus liquid test ending generally 7 day time of needs about the 7 days holding time that virus liquid can meet needs of production more than 0 DEG C, check satisfactory virus liquid just can be used for the vaccine that manufactures a finished product), especially under 0 DEG C ~ 8 DEG C conditions, can at least preserve 28 days, virus titer declines within 1 titre.And preserve below 0 DEG C, when 7 days First Determination virus titers, more than 1 titre that just declines, and afterwards preservation 14 days, 21 days and 28 days when measuring virus titer, very nearly the same, substantially more stable with tiring when preserving 7 days.Analyze its reason, may be that virus liquid can be freezing when preserving below 0 DEG C, freeze thawing virus liquid can cause the death of fractionated viral, causes virus titer to reduce.
In order to verify above-mentioned possible reason further, devising temperature conditions below 0 DEG C and preserving and multigelation virus liquid, measuring it at different storage life virus titer.
Table 3 less than 0 DEG C condition multigelation virus liquid storage life
As can be seen from Table 3, virus liquid, after multigelation, is tired and is declined obviously, and freeze thawing is serious on virus activity impact.
The preparation of embodiment 5 vaccine
Prepared by embodiment 3 virus liquid B1 be divided into 4 parts, add respectively embodiment 1 prepare stabilizing agent N1, N2, N3 and N4, fully shake up, get product after carrying out lyophilisation rapidly Y1, Y2, Y3 and Y4.
Carry out product inspection according to " People's Republic of China's veterinary drug allusion quotation " (version in 2010), meet the regulation of " pseudorabies living vaccines ", every part vaccine is containing virus>=10
6tCID
50, Y1, Y2, Y3 and Y4 every part vaccine is respectively 10 containing virus
6.59tCID
50, 10
6.6tCID
50, 10
6.54tCID
50with 10
6.52tCID
50.
The storage life test of embodiment 6 vaccine
Finished product vaccine Y1, Y2, Y3 and Y4 of embodiment 5 being prepared, preserve under being placed in 100 DEG C, 37 DEG C, 2 DEG C ~ 8 DEG C conditions respectively, and different time samples, and measures viral level and physical behavior thereof.
Meanwhile, virus liquid B1 prepared by Example 3, adds the stabilizing agent containing 5% lactose, 10% skim milk aqueous solution, fully shakes up, quantitative separating, namely obtain vaccine finished product D after carrying out lyophilisation rapidly.Carry out product inspection according to " People's Republic of China's veterinary drug allusion quotation " (version in 2010), meet the regulation of " pseudorabies living vaccines ", every part vaccine is containing virus>=10
6tCID
50, every part vaccine is respectively 10 containing virus
6.5tCID
50.By vaccine D in contrast, preserve under being placed in 100 DEG C, 37 DEG C, 2 DEG C ~ 8 DEG C conditions, different time samples, and measures viral level and physical behavior thereof.
100 DEG C, table 4 boils physical behavior and the virus titer mensuration of 10 minutes indirectly
Storage life test under table 5 37 DEG C of conditions
Storage life test under table 62 DEG C ~ 8 DEG C conditions
Can find out that porcine pseudorabies freeze-dried live vaccine of the present invention is more than 0 DEG C and significant stability by above data, especially under 2 DEG C ~ 8 DEG C conditions, its stability can keep 36 months, even if under 37 DEG C of conditions, also 10 days can be kept, its decline of tiring is no more than 0.5 titre, shows its significant stability.The restriction that further breakthrough " cold chain " transport is preserved, has greatly saved cost, has also more been conducive to applying of porcine pseudorabies freeze-dried live vaccine of the present invention.
The preparation of embodiment 7 diluent
The composition of diluent and constituent content are in table 7.
The composition content of table 7 diluent
| Group | Sorbitol (W/V) | Sodium thiosulfate (W/V) | Water for injection (V/V) |
| X1 | 1% | 1% | The amount of supplying 100% |
| X2 | 1.5% | 3.5% | The amount of supplying 100% |
| X3 | 3% | 5% | The amount of supplying 100% |
For often kind of diluent, take sodium thiosulfate and be dissolved in 50ml water for injection, then add sorbitol, be settled to 100ml.0.22 μm of membrane filtration, 100 DEG C of sterilizings 30 minutes.
Embodiment 8 diluent is on the impact of pseudorabies cytotoxic activity
Freeze dried vaccine prepared by diluent embodiment 7 prepared and embodiment 5 and embodiment 6 mixes, and places 30 minutes, 2 hours, 6 hours, 12 hours and 24 hours respectively, measure its TCID under putting room temperature (25 DEG C) environment
50change.Arranging normal saline group is matched group.
Table 8 diluent is on the impact of pseudorabies cytotoxic activity
As can be seen from Table 8, can keep the long period at normal temperatures after diluted vaccine of the present invention, 24h vaccine valence is declined by less than 0.5 titre, far below the holding time of customary physiological saline as diluent.And find unexpectedly, carry out mixing and mixing with diluent of the present invention with vaccine prepared by conventional stabilizer with normal saline diluent with adding the vaccine of stabilizing agent of the present invention, obtain goods stability be also greater than the stability of conventional stabilizer vaccine with normal saline diluent mixing resulting product.Analyze its reason, may with the present invention's stabilizer element used and diluent ingredient relevant, ensure that the stability of vaccine.
The immunity test of embodiment 9 pseudorabies live vaccine
21 age in days PRV negative antibody piglets 16 are divided into 4 groups at random, according to table 9 immune vaccine 1 part, the isopyknic vaccine diluent of matched group immunity.Within latter 28 days, counteracting toxic substances dosage is 10 with PRV (Pseudorabies virus) Fa strain counteracting toxic substances in immunity
7.0pFU, adopts nasal drip.After counteracting toxic substances, every day measures piglet body temperature, observes clinical symptoms and death condition.
Table 9 Study On Immunogenicity animal divides into groups
| Group | Immune vaccine | Immunizing dose |
| 1 group | Y1+X1 | 10 6.59TCID 50/ head part |
| 2 groups | Y2+X2 | 10 6.6TCID 50/ head part |
| 3 groups | Y3+X3 | 10 6.54TCID 50/ head part |
| 4 groups | Y4+X2 | 10 6.52TCID 50/ head part |
| Matched group | X2 | With immune group equal-volume |
After vaccine immunity, weekly with reference to the NAT of the method mensuration vaccine group of GB/T18641-2002 method serum neutralization test, the results are shown in Table 10.
The antibody situation of different time after table 10 pseudorabies live vaccine immunity piglet
The result display of table 10, after pseudorabies live vaccine immunity piglet, can produce higher neutralizing antibody, and antibody horizontal raises gradually with immunization time.
Immunity is counteracting toxic substances after latter 28 days, and counteracting toxic substances strain is porcine pseudorabies virus Fa strain, and counteracting toxic substances dosage is 10
7.0pFU, observation clinical symptoms and death condition are in table 11, and after counteracting toxic substances, every day measures piglet body temperature in table 12.
Counteracting toxic substances situation after table 11 pseudorabies live vaccine immunity piglet
Mean body temperature change after the piglet counteracting toxic substances of table 12 pseudorabies vaccine immunity
The result display of table 11 and table 12; after pseudorabies live vaccine immunity piglet; although (occurring clinical symptoms) can not be infected by blocking virus; but 100%(4/4 can be provided for piglet) protection; and to contrast after piglet counteracting toxic substances 4 days all dead afterwards; therefore, pseudorabies live vaccine has good protection.
The above is only the preferred embodiments of the present invention, not any pro forma restriction is done to the present invention, although the present invention discloses as above with preferred embodiment, but and be not used to limit the present invention, any those skilled in the art, not departing from the scope of technical solution of the present invention, make a little change when the technology contents of above-mentioned announcement can be utilized or be modified to the Equivalent embodiments of equivalent variations, in every case be the content not departing from technical solution of the present invention, according to any simple modification that technical spirit of the present invention is done above embodiment, equivalent variations and modification, all still belong in the scope of technical solution of the present invention.
Claims (10)
1. produce a method for pseudorabies living vaccines with subculture cell source, it is characterized in that, comprise the steps:
(1) pseudorabies virus low virulent strain is cultivated with passage cell;
(2) virus liquid is gathered in the crops;
(3) add stabilizing agent, obtain subculture cell source pseudorabies disease live-vaccine through lyophilisation.
2. a kind of subculture cell source according to claim 1 produces the method for pseudorabies living vaccines, and it is characterized in that, described step (1) comprises the steps:
(1a) the going down to posterity and cultivation of seedling cell: described passage cell, through pancreatin cell dispersal had digestive transfer culture, continues to cultivate with cell growth medium, forms passage cell monolayer;
(1b) breeding of cell seed culture of viruses: described pseudorabies virus low virulent strain is seeded to the passage cell monolayer obtained in described step (1a), continues to cultivate with cell maintenance medium; After 24h ~ 44h, harvesting cultivates virus liquid as production seed culture of viruses;
(1c) seedling venom breeding: get the passage cell monolayer obtained in described step (1a), discard described cell growth medium, inoculate the production seed culture of viruses obtained in described step (1b), continues to cultivate.
3. a kind of subculture cell source according to claim 2 produces the method for pseudorabies living vaccines, it is characterized in that, the temperature used of the cell culture in described step (1a), (1b), (1c) is 36 DEG C ~ 38 DEG C; Inoculum concentration during inoculation in described step (1b), (1c) is the maintenance medium of the Cells for production seed culture of viruses of 1% ~ 2% percent by volume; Cell growth medium in described step (1a) is containing the MEM liquid of 90% ~ 97% percent by volume and the hyclone of 3% ~ 10% volume ratio, and the pH value of described cell growth medium is 7.0 ~ 8.0; Cell maintenance medium in described step (1b), (1c) is containing the MEM liquid of 95% ~ 99% percent by volume and the hyclone of 1% ~ 5% volume ratio, and the pH value of described cell maintenance medium is 7.1 ~ 7.5.
4. a kind of subculture cell source according to claim 1 produces the method for pseudorabies living vaccines, it is characterized in that, after described step (2) comprises the steps: to connect poison, 24h ~ 44h harvesting cultivates venom, and the venom of results is placed in more than 0 DEG C preservation.
5. a kind of subculture cell source according to claims 1 to 4 produces the method for pseudorabies living vaccines, and it is characterized in that, described passage cell is pig testis subculture cells ST, porcine kidney cell PK15 or porcine kidney cell IBRS-2.
6. a pseudorabies living vaccines, is characterized in that, described pseudorabies living vaccines is obtained by the method described in Claims 1 to 4.
7. pseudorabies living vaccines according to claim 6, is characterized in that, described stabilizing agent comprises trehalose, L-sodium and vitamin C.
8. pseudorabies living vaccines according to claim 7, it is characterized in that, the ratio that described trehalose accounts for stabilizing agent is 4% ~ 6%W/V, and the ratio that described L-sodium accounts for stabilizing agent is 4% ~ 6%W/V, and the ratio that described vitamin C accounts for stabilizing agent is 0.1% ~ 0.3%W/V.
9. pseudorabies living vaccines according to claim 7, is characterized in that, described stabilizing agent comprises polyvinylpyrrolidone further; The ratio that described polyvinylpyrrolidone accounts for stabilizing agent is 4% ~ 6%W/V.
10. pseudorabies living vaccines according to claim 6, is characterized in that, it comprises diluent further; Described diluent comprises sorbitol and sodium thiosulfate; The ratio that described sorbitol accounts for diluent is 1% ~ 3%W/V, and the ratio that described sodium thiosulfate accounts for diluent is 1% ~ 5%W/V.
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| CN106310248A (en) * | 2015-06-18 | 2017-01-11 | 普莱柯生物工程股份有限公司 | Purpose of porcine pseudorabies virus live vaccine composition used for treating porcine pseudorabies |
| CN106310248B (en) * | 2015-06-18 | 2019-12-20 | 普莱柯生物工程股份有限公司 | Application of porcine pseudorabies virus live vaccine composition in treating porcine pseudorabies |
| CN105602909A (en) * | 2016-03-29 | 2016-05-25 | 四川海林格生物制药有限公司 | Method for culturing porcine pseudorabies virus |
| CN105624123A (en) * | 2016-03-29 | 2016-06-01 | 四川海林格生物制药有限公司 | Cell affinity agent for improving porcine pseudorabies virus in-vitro infection efficiency and use method thereof |
| CN107224579A (en) * | 2017-06-09 | 2017-10-03 | 广东渔跃生物技术有限公司 | A kind of method that pseudorabies live vaccine is produced with continuous cell line |
| CN107224579B (en) * | 2017-06-09 | 2019-09-06 | 广州渔跃生物技术有限公司 | A method of pseudorabies live vaccine is produced with continuous cell line |
| CN110241090A (en) * | 2019-05-07 | 2019-09-17 | 江苏南农高科技股份有限公司 | A kind of method of full suspension cell culture production porcine pseudorabies virus antigen |
| CN110241090B (en) * | 2019-05-07 | 2023-10-13 | 江苏南农高科技股份有限公司 | Method for producing porcine pseudorabies virus antigen by full suspension cell culture |
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