CN105521487A - Method for preparing pseudorabies live vaccines from DF1 continuous cells and product prepared by method - Google Patents
Method for preparing pseudorabies live vaccines from DF1 continuous cells and product prepared by method Download PDFInfo
- Publication number
- CN105521487A CN105521487A CN201410515820.4A CN201410515820A CN105521487A CN 105521487 A CN105521487 A CN 105521487A CN 201410515820 A CN201410515820 A CN 201410515820A CN 105521487 A CN105521487 A CN 105521487A
- Authority
- CN
- China
- Prior art keywords
- cell
- pseudorabies
- described step
- passage
- produce
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention provides a method for preparing pseudorabies live vaccines from DF1 continuous cells and a product prepared by the method. The method includes the steps of culturing a pseudorabies virus attenuated strain by the DF1 continuous cells; obtaining cell culture virus fluid; adding stabilizers and antibiotics and obtaining the pseudorabies DF1 live vaccines after vacuum freeze drying. The method has the advantages that the DF1 continuous cells which are heterogenous continuous cells are used for culturing pseudorabies attenuated viruses, so that the possibility that homologous cells have unknown swine-source microorganisms can be avoided and pure vaccines can be better guaranteed.
Description
Technical field
The present invention relates to a kind of method and the goods thereof that utilize DF1 passage cell production pseudorabies living vaccines, belong to biomedicine technical field.
Background technology
It is chick embryo fibroblast primary cell that China produces pseudorabies living vaccines cell used at present.With chick embryo fibroblast primitive cell culture pseudorabies virus, it is not high to produce malicious titre; The manufacture of chick embryo fibroblast primary cell is because often criticizing the raw-material difference of Embryo Gallus domesticus, and cell differences between batches are larger; Chick embryo fibroblast primary cell cell debris and foreign protein many, Pigs Inoculated easily causes allergic reaction.
Summary of the invention
In view of this, the object of this invention is to provide a kind of DF1 passage cell that utilizes overcoming above-mentioned defect and produce the method for pseudorabies living vaccines.
For achieving the above object, the invention provides a kind of method utilizing DF1 passage cell to produce pseudorabies living vaccines, comprising the steps:
(1) pseudorabies virus low virulent strain is cultivated with DF1 passage cell;
(2) the cell culture venom that described step (1) obtains is gathered in the crops;
(3) add stabilizing agent and antibiotics in the cell culture venom obtained in described step (2), obtain pseudorabies DF1 cell live vaccine through lyophilisation.
Further, rolling bottle or bioreactor is used to cultivate in wherein said method.
Further, wherein said step (1) comprises the steps:
(1a) the going down to posterity and cultivation of seedling cell: described passage cell, through EDTA-pancreatin cell dispersal liquid had digestive transfer culture, continues to cultivate with cell growth medium, forms DF1 passage cell monolayer;
(1b) breeding of cell seed culture of viruses: described pseudorabies virus low virulent strain is seeded to the DF1 passage cell monolayer obtained in described step (1a), continues to cultivate with cell maintenance medium; After 2 ~ 3 days, harvesting cultivates venom as production seed culture of viruses;
(1c) breeding of seedling venom: get the DF1 passage cell monolayer obtained in described step (1a), discard described cell growth medium, inoculation, containing the cell maintenance medium of the seed culture of viruses obtained in described step (1b), continues to cultivate.
Further, the cell seed culture of viruses that wherein said step (1b) obtains, every 1ml is containing virus>=10
8tCID
50.
Further, the temperature used of the cell culture in wherein said step (1a), (1b), (1c) is 36 DEG C ~ 37 DEG C.
Further, the inoculum concentration in wherein said step (1b), (1c) during inoculation by volume percentages is the maintenance medium containing 1% ~ 2% Cells for production seed culture of viruses.
Further, cell growth medium in wherein said step (1a) is containing 90% ~ 92%MEM liquid, 8% ~ 10% calf serum and 100 ~ 200 units/ml mycillin, the pH value of described cell growth medium is 7.0 ~ 7.2, and wherein the unit of MEM liquid and calf serum is percent by volume.
Further, cell maintenance medium in wherein said step (1b) and (1c) is containing 95% ~ 98%MEM liquid, 2% ~ 5% calf serum and 100 ~ 200 units/ml mycillin, the pH value of described cell maintenance medium is 7.2 ~ 7.4, and wherein the unit of MEM liquid and calf serum is percent by volume.
Further, after wherein said step (2) comprises the steps: to connect poison, harvesting on the 2nd ~ 3 cultivates venom, and less than-20 DEG C preservations put by the venom of results.
Further, the venom that wherein said step (2) is gathered in the crops, every 1ml is containing virus>=10
8tCID
50;
Further, the pseudorabies DF1 cell live vaccine that wherein said step (3) obtains, every part vaccine is containing virus>=10
6tCID
50.
In addition, the present invention also provides a kind of pseudorabies living vaccines, and described pseudorabies living vaccines is obtained by said method.
The technique effect that the present invention realizes is as follows:
1, utilize DF1 passage cell to produce pseudorabies living vaccines, have production technology simple and stable, easy to operate, viral level is high, and differences between batches are little, easy to control the quality, can significantly improve vaccine seed output and quality, reduces the advantages such as anaphylaxis;
2, the pseudorabies living vaccines safety utilizing the present invention to produce is good, immune efficacy is high, has good immanoprotection action to pseudorabies strong virus attack;
3, utilize DF1 passage cell to cultivate the weak poison of pseudorabies virus, because DF1 passage cell is allos passage cell, the possibility of the unknown pig source microorganism of homologous cell band can be avoided, more can ensure the pure of vaccine.
Accompanying drawing explanation
The production technological process of Fig. 1: pseudorabies DF1 passage cell live vaccine.
Fig. 2: the serum antibody Fluctuation figure of immunity inoculation pig.
Detailed description of the invention
For making the present invention easier to understand, set forth the present invention further below in conjunction with specific embodiment.Should be understood that these embodiments only for illustration of the present invention instead of for limiting the scope of the invention, NM specific experiment method in the following example, conveniently experimental technique carries out usually.The seed culture of viruses that the genetic origin pseudorabies DF1 passage cell live vaccine of biomaterial of the present invention uses is pseudorabies virus Bartha strain, purchased from Chinese veterinary microorganism culture presevation administrative center (CVCC).The cell that pseudorabies DF1 passage cell live vaccine of the present invention uses is chick embryo fibroblast passage cell (DF1 passage cell), purchased from Chinese veterinary microorganism culture presevation administrative center (CVCC).Pseudorabies DF1 passage cell live vaccine inspection of the present invention is Pseudorabies virus Ma strain with strong poison, purchased from Chinese veterinary microorganism culture presevation administrative center (CVCC).
Iting is noted that following illustrating is all exemplary, being intended to the invention provides further instruction.Except as otherwise noted, all Science and Technology terms used herein have the identical meanings usually understood with the technical field of the invention personnel.
The preparation of embodiment 1 pseudorabies living vaccines
As shown in Figure 1, a kind of method utilizing DF1 passage cell to produce pseudorabies living vaccines, comprises the steps:
(1) seedling cell is selected: DF1 passage cell;
(2) the going down to posterity and cultivation of seedling cell: above-mentioned DF1 passage cell, through EDTA-pancreatin cell dispersal liquid had digestive transfer culture, continuing to cultivate with cell growth medium, when forming good monolayer, going down to posterity or virus inoculation for continuing;
Cell culture go down to posterity and in daily maintenance process, a large amount of expending all is needed in cultivation utensil, culture fluid and various preparation, and cell starts original cuiture once leave live body, its various biological natures all will change gradually and constantly have new change along with the increase of passage number and the change of vitro condition.Therefore cell cryopreservation is carried out in time very necessary.Therefore the DF1 passage cell of above-mentioned cultivation is carried out cell cryopreservation, need cell recovery be carried out time to be used.
Cell cryopreservation: select the cell being in exponential phase, cells frozen storing liquid (10% ~ 85%MEM solution, 10% ~ 90% hyclone, 5% ~ 10%DMSO) is added after being disperseed by cell dissociation by above-mentioned passage method, be sub-packed in cell cryopreservation tube, with sealing compound sealing, labeled cell title, generation, date.First through ladder cooling (with the speed of-1 ~-3 DEG C/min cooling), finally move in liquid nitrogen (-196 DEG C).
Cell recovery: take out freeze-stored cell in liquid nitrogen, put into rapidly 40 DEG C of warm water, slowly shake is to melting completely, centrifugal (centrifugal 5 ~ 10 minutes of 1000r/min) the supernatant that inclines, add 5 ~ 7ml (T25 bottle) nutritional solution dispersion suspension cell, proceed to culture bottle, put 36 ~ 37 DEG C and continue to cultivate, grow up to good monolayer.
(3) breeding of cell seed culture of viruses: by the well-grown above-mentioned DF1 passage cell monolayer of pseudorabies virus low virulent strain inoculation, adds cell maintenance medium and continues to cultivate; After 2 ~ 3 days, harvesting cultivates venom as production seed culture of viruses;
Cell seed culture of viruses is identified: the qualification of cell seed culture of viruses meets pseudorabies virus low virulent strain seed culture of viruses standard completely, has no side effect safely to pig, and the every 1ml of cell seed culture of viruses is containing virus>=10
8tCID
50.
(4) breeding of seedling venom: get the DF1 passage cell culture bottle forming good monolayer, discard cell growth medium, inoculate above-mentioned toxic maintenance medium, continues to cultivate; After connecing poison, harvesting on the 2nd ~ 3 cultivates venom, and less than-20 DEG C preservations put by the venom of results;
The inspection of seedling venom: test by existing " People's Republic of China's veterinary drug allusion quotation " annex, should without antibacterial, mycete, mycoplasma growth.Cell venom has no side effect safely to pig, and each time virus-culturing fluid of receiving is tired with raji cell assay Raji respectively, and every 1ml is containing virus>=10
8tCID
50;
(5) join Seedling, subpackage and lyophilizing: by the virus-culturing fluid be up to the standards, be mixed in same container, add stabilizing agent, add antibiotic simultaneously, fully shake up, quantitative separating; Get product after carrying out lyophilisation rapidly after subpackage.
Product inspection: test by existing " People's Republic of China's veterinary drug allusion quotation ", should meet the regulation of " pseudorabies living vaccines ", have no side effect safely to pig, every part vaccine is containing virus>=10
6tCID
50.
Wherein, involved in each step actual conditions is as follows:
Cell culture in described step (2), (3), (4) temperature used is 36 DEG C ~ 37 DEG C.
Described step (3), inoculum concentration in (4) during inoculation are 1% ~ 2%(ml/ml) maintenance medium of Cells for production seed culture of viruses.
The formula of the cell growth medium in described step (2) is: 90% ~ 92%(ml/ml) MEM liquid, 8% ~ 10%(ml/ml) calf serum add 100 ~ 200 units/ml mycillin, pH value is adjusted to 7.0 ~ 7.2.
The formula of the cell maintenance medium in described step (3), (4) is: 95% ~ 98%(ml/ml) MEM liquid, 2% ~ 5%(ml/ml) calf serum add 100 ~ 200 units/ml mycillin, pH value is adjusted to 7.2 ~ 7.4.
Embodiment 2 pseudorabies DF1 passage cell live vaccine is to the safety testing of pig
1 materials and methods
1.1 test vaccines
The pseudorabies DF1 passage cell live vaccine 3 batches of laboratory development, lot number is respectively DF201301, DF201302, DF201303.
1.2 test animal
Derive from the health pig of Guangdong Winsun Bio-Pharmaceutical Co., Ltd.'s laboratory animal field from the pseudorabies virus neutralizing antibody feminine gender of numerous autotrophy.
The safety test of 1.3 pairs of pigs
1.3.1 the safety test of a single dose immunization inoculation
Choose the health pig of 20 ~ 25 ages in days, 50 ~ 55 ages in days, 3 batches of Lab Products are carried out single dose immunization inoculation to the pig of different days, often criticizes vaccine immunity 5 pigs respectively, every pig muscle injection 1 part vaccine, after inoculation, every day at upper and lower noon each thermometric observes 1 time, observes 14.And set up negative control pig 5 respectively, and raise thermometric under immune swine the same terms and observe.
Choose the sow of conceived 82 ~ 94 days, 3 batches of Lab Products are carried out single dose immunization inoculation to it, often criticize vaccine immunity 5 pigs respectively, every pig muscle injection 1 part vaccine, morning and afternoon every day, each thermometric observed 1 time.Observe immune sow spirit, appetite, body temperature situation follow the tracks of the condition of production.And set up blank negative control pregnancy sow on the 82nd ~ 94 5, and raise thermometric under immune sow the same terms and observe the condition of production.
1.3.2 single dose repeats the safety test of immunity inoculation
Choose the health pig of 20 ~ 25 ages in days, 50 ~ 55 ages in days, 3 batches of Lab Products are carried out single dose to the pig of different days and repeats immunity inoculation, often criticize vaccine immunity 5 pigs respectively, every pig muscle injection 1 part vaccine, after inoculation, every day at upper and lower noon each thermometric observes 1 time, inoculate latter 14 days and carry out repeated inoculation, observe 14 after repeated inoculation.And set up negative control pig 5 respectively, raise thermometric with under immune swine the same terms.
Choose the sow of conceived 68 ~ 80 days, 3 batches of Lab Products are carried out single dose to it and repeats immunity inoculation, often criticize vaccine immunity 5 pigs respectively, every pig muscle injection 1 part vaccine, after inoculation, every day at upper and lower noon each thermometric observes 1 time, inoculate and carry out repeated inoculation after 14 days, observe immune sow spirit, appetite, body temperature situation and the condition of production.And set up blank negative control pregnancy sow on the 68th ~ 80 5, and raise thermometric under immune swine the same terms and observe the condition of production.
1.3.3 the safety test of overdose immunity inoculation
Choose the health pig of 20 ~ 25 ages in days, 50 ~ 55 ages in days, 3 batches of Lab Products are carried out overdose immunity inoculation to the pig of different days, often criticizes vaccine immunity 5 pigs respectively, every pig muscle injection 10 part vaccines, observe 14 after inoculation, morning and afternoon every day, each thermometric observed 1 time.And set up negative control pig 5 respectively, and raise thermometric under immune swine the same terms and observe.
Choose the sow of conceived 82 ~ 94 days, 3 batches of Lab Products are carried out overdose immunity inoculation, often criticize vaccine immunity 5 pigs respectively, every pig muscle injection 10 part vaccines, every day at the upper and lower noon, each thermometric observed 1 time.Observe immune sow spirit, appetite, body temperature situation and the condition of production.And set up blank negative control pregnancy sow on the 82nd ~ 94 5, and raise thermometric under immune sow the same terms and observe the condition of production.
Except general safety inspection routinely, also to injection site after inoculation until do with or without color changes such as skin rubefaction before off-test, formed with or without swelling and lump, near inoculation position lymph node with or without the observation inspection of the pathological changes such as enlargement.
2 results
The safety test result of 2.1 single dose immunization inoculations
By testing with 3 batches of vaccines to the health pig of 20 ~ 25 ages in days, 50 ~ 55 ages in days and conceived sow on the 82nd ~ 94, through a single dose immunization inoculation, all without any untoward reaction, to pig safety, to pig safety, pig that in-pig farrows is all healthy to survive.
2.2 single doses repeat the safety test result of immunity inoculation
By testing with 3 batches of vaccines to the health pig of 20 ~ 25 ages in days, 50 ~ 55 ages in days and conceived in-pig on the 68th ~ 80, through 2 single dose immunization inoculations, all without any untoward reaction, to pig safety, survive to pig that in-pig farrows is all healthy.
The safety test result of 2.3 overdose immunity inoculations
Test 3 batches of vaccines are carried out overdose immunity inoculation to the health pig of 20 ~ 25 ages in days, 50 ~ 55 ages in days and conceived sow on the 82nd ~ 94.Pigs Inoculated observes 14, and spirit, appetite etc. are normal, and after inoculating front and inoculation, body temperature normally, all without any untoward reaction, to pig safety, to the whole healthy survival of pig that in-pig farrows.
Except above safety inspection is routinely all qualified, all pigs until the viewing duration of off-test, are showed no skin rubefaction after injection, and local touches without swelling and lump, and submandibular lymph nodes is without situations such as enlargements.At the end of experiment, local, injection site is not seen to the dissection of part Pigs Inoculated yet and occur the pathological changes such as swelling with contiguous lymph node.Prove that pseudorabies DF1 passage cell live vaccine is safe to pig.
3 conclusions
3.1 test with 3 batches of pseudorabies DF1 passage cell live vaccine 20 ~ 25 ages in days, the health pig of 50 ~ 55 ages in days and the sow of latter half of gestation, intramuscular injection is adopted to carry out immunity inoculation respectively, no matter be that single dose one shot immunity or single dose repeat immunity inoculation, all have no adverse reaction; Adopt overdose immunity inoculation, the health pig of 20 ~ 25 ages in days, 50 ~ 55 ages in days and sow heavy in pig spirit, appetite, body temperature, production status are all normal.Result shows, no matter pseudorabies DF1 passage cell live vaccine, to the immunity inoculation of different days pig and sow heavy in pig, is single dose, overdose, still once or repeated inoculation, is all safe.
3.2 result of the tests show, pseudorabies DF1 passage cell live vaccine takes piglet and the sow heavy in pig of the method difference intramuscular injection different days such as single dose, single dose repetition and an overdose, all Pigs Inoculateds all have no adverse reaction, pig that in-pig farrows is healthy survival all, all not there are abnormal conditions, illustrate can utilization and extention DF1 passage cell produce pseudorabies disease live-vaccine.In addition, DF1 passage cell produce pseudorabies disease live-vaccine (DF1-1, DF1-2, DF1-3) immune health pig after 7 days antibody horizontal start rise, peak after 21 days, high-caliber immune antibody can maintain the long period, until immunity latter 365 days serum antibodys still remain positive.Illustrate that the pseudorabies disease live-vaccine immune swine antibody positive that DF1 passage cell is produced can maintain 12 months, as shown in Figure 2.Annotation: in figure, serum antibody is pseudorabies vaccines poison antibody, is greater than 0.70 for negative, is less than or equal to 0.60 for positive, between be suspicious.
Embodiment 2 pseudorabies DF1 passage cell live vaccine produces poison test
1 material
1.1 seeds culture of viruses are provided by Guangdong Winsun Bio-Pharmaceutical Co., Ltd..
1.2 cells are provided by Guangdong Winsun Bio-Pharmaceutical Co., Ltd..
2 methods
From working cell storehouse recovery cell and by passage, get form good monolayer F10, F15, F20, F25, F30 for cell, discard nutritional solution, inoculation is containing 0.1% ~ 3%(ml/ml) maintenance medium of production seed culture of viruses, put 36 ~ 37 DEG C to continue to cultivate, after connecing poison, every day observes twice, and gather in the crops virus liquid when CPE reaches 50% ~ 100%, less than-20 DEG C preservations put by the venom of results.The cell venom getting each generation working cell results carries out viral level mensuration respectively.And carry out double repeated experiment.
3 results
From the cell of working cell storehouse recovery, reaching F40 cell still can well-grown, observation of cell form and vigor no abnormality seen under low power lens.With F10, F15, F20, F25, F30 cell culture pseudorabies virus vaccine strain cultured cells venom, measure through viral level.Result shows: F10, F15, F20, F25, F30 for the viral level no significant difference of each generation cell culture pseudorabies virus vaccine strain institute harvesting venom, viral level>=10
8.0tCID
50/ ml.
Embodiment 3DF1 passage cell mutagenicity test
1 material
1.1 cell
DF1-F3, F20, F30; C6-F18; NIH3T3 cell.Thered is provided by Guangdong Winsun Bio-Pharmaceutical Co., Ltd..
1.2 nude mices: male, 3 ~ 4 week age, purchased from Shanghai Slac Experimental Animal Co., Ltd..
2 methods
2.1 cell colonies form test
Preparation DMEM and low melting-point agarose liquid, low melting-point agarose liquid maintains 40 DEG C makes it not solidify.Prepare bottom-layer agar in 1:1 ratio mixing agarose and DMEM, get 3ml and inject diameter 6cm plate, to be cooledly to solidify; Peptic cell makes cell suspension, counting, inoculating cell; Mix mutually in sterile tube and prepare top agar, then add the cell suspension of 0.2ml in pipe, fully mixing, injection have been covered with agarose bottom plate, then form pair agar layer.After top-layer agar solidifies, insert in 37 DEG C of CO2 incubators and cultivate 25 days.Every strain cell inoculates 10 wares, counts to there being the clone of more than 50 cells.
2.2 nude mice Tumorigenesis
Get experimental cell and the negative control cell of above-mentioned exponential phase respectively, through digestion, counting, dilution after PBS washing; To take the logarithm the C6 cell of trophophase, through digestion, counting, dilution after PBS washing.Get 200 μ l cell suspension subcutaneous injection after the sterilization of nude mice nape portion local skin respectively, routine observation.After disconnected neck puts to death nude mice, get tumor tissue immediately, fix through 10% formalin, make paraffin section.Carry out HE dyeing according to a conventional method, basis of microscopic observation.
2.3 karyologys detect
Auxocyte of taking the logarithm adds colchicine0.2ug/ml culture fluid, 37 DEG C of 3-7 hour.DHank ' s washes once, and 0.25%trypsin digests, and DHank ' s washes once, 0.075MKCl37 DEG C hypotonic 15 minutes, methanol-acetic acid is fixed 3-4 time, centrifugal rear a small amount of fixative suspension cell, drips sheet, dried overnight, Giemsa dyes 10 minutes, and washing is air drying also, and dimethylbenzene is transparent, mounting, observes the Chromosome number that 50 are in the cell of mitosis metaphase.
3 results
3.1 cell colonies form result of the test
Cultivated through 25 days, DF1 (F3), DF1 (F20), DF1 (F30) and negative control NIH3T3 cell all do not form colony, and only at upper layer growth, and C6 cell can be observed the colony that differs in size under microscope mirror at the 12nd day.
3.2 nude mice Tumorigenesis results
Observed through 21 days after cell inoculation, the nude mice of DF1 (F3), DF1 (F20), DF1 (F30) and negative control cell NIH3T3 inoculation does not all find the formation of tuberosity or tumor; Latter 22 days of cell inoculation, the nude mice of getting wherein half carries out cuing open inspection, observes the formation that each lymph node and organa parenchymatosum all do not find tuberosity or tumor; Half nude mice cuts open inspection after observing 90 days in addition, observes the formation that each lymph node and organa parenchymatosum all do not find tuberosity or tumor, is also formed without transplanted tumor.And the nude mice of C6 cell inoculation, in the subcutaneous tumor can observing soybean grain size of its nape after about two weeks, and along with the prolongation of time, tumor block becomes large gradually.Get this tumor tissue, visible tumor tissue matter is tough, tangent plane is greyish white flesh of fish sample.Fix through formalin, HE dyeing after paraffin section, rich blood vessel in visible tumor tissue under mirror, oncocyte is fusiformis, arrangement disorder, and karyokinesis is vigorous, with special-shaped core and pathologic mitosis.
3.3 karyology testing results: Chromosome number is no abnormal.
4 conclusions
This time the soft agarose test of inspected DF1 (F3), DF1 (F20) and DF1 (F30) cell is for negative, and nude mouse tumor forms test for negative, and Chromosome number is no abnormal.
The above, be only the preferred embodiments of the present invention, should be understood that; for the those of ordinary skill in this technology; under the prerequisite not departing from core technical features of the present invention, can also make some improvements and modifications, these retouchings and improvement also should belong to scope of patent protection of the present invention.
Claims (12)
1. utilize DF1 passage cell to produce a method for pseudorabies living vaccines, it is characterized in that, comprise the steps:
(1) pseudorabies virus low virulent strain is cultivated with DF1 passage cell;
(2) the cell culture venom that described step (1) obtains is gathered in the crops;
(3) add stabilizing agent and antibiotics in the cell culture venom obtained in described step (2), obtain pseudorabies DF1 cell live vaccine through lyophilisation.
2. utilize DF1 passage cell to produce the method for pseudorabies living vaccines as claimed in claim 1, it is characterized in that, in described method, use rolling bottle or bioreactor to cultivate.
3. utilize DF1 passage cell to produce the method for pseudorabies living vaccines as claimed in claim 1, 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 EDTA-pancreatin cell dispersal liquid had digestive transfer culture, continues to cultivate with cell growth medium, forms DF1 passage cell monolayer;
(1b) breeding of cell seed culture of viruses: described pseudorabies virus low virulent strain is seeded to the DF1 passage cell monolayer obtained in described step (1a), continues to cultivate with cell maintenance medium; After 2 ~ 3 days, harvesting cultivates venom as production seed culture of viruses;
(1c) breeding of seedling venom: get the DF1 passage cell monolayer obtained in described step (1a), discard described cell growth medium, inoculation, containing the cell maintenance medium of the seed culture of viruses obtained in described step (1b), continues to cultivate.
4. utilize DF1 passage cell to produce the method for pseudorabies living vaccines as claimed in claim 3, it is characterized in that, the cell seed culture of viruses that described step (1b) obtains, every 1ml is containing virus>=10
8tCID
50.
5. utilize DF1 passage cell to produce the method for pseudorabies living vaccines as claimed in claim 3, it is characterized in that, the temperature used of the cell culture in described step (1a), (1b), (1c) is 36 DEG C ~ 37 DEG C.
6. utilize DF1 passage cell to produce the method for pseudorabies living vaccines as claimed in claim 3, it is characterized in that, the inoculum concentration in described step (1b), (1c) during inoculation by volume percentages is the maintenance medium containing 1% ~ 2% Cells for production seed culture of viruses.
7. utilize DF1 passage cell to produce the method for pseudorabies living vaccines as claimed in claim 3, it is characterized in that, cell growth medium in described step (1a) is containing 90% ~ 92%MEM liquid, 8% ~ 10% calf serum and 100 ~ 200 units/ml mycillin, the pH value of described cell growth medium is 7.0 ~ 7.2, and wherein the unit of MEM liquid and calf serum is percent by volume.
8. utilize DF1 passage cell to produce the method for pseudorabies living vaccines as claimed in claim 3, it is characterized in that, cell maintenance medium in described step (1b) and (1c) is containing 95% ~ 98%MEM liquid, 2% ~ 5% calf serum and 100 ~ 200 units/ml mycillin, the pH value of described cell maintenance medium is 7.2 ~ 7.4, and wherein the unit of MEM liquid and calf serum is percent by volume.
9. utilize DF1 passage cell to produce the method for pseudorabies living vaccines as claimed in claim 1, it is characterized in that, after described step (2) comprises the steps: to connect poison, harvesting on the 2nd ~ 3 cultivates venom, and less than-20 DEG C preservations put by the venom of results.
10. utilize DF1 passage cell to produce the method for pseudorabies living vaccines as claimed in claim 9, it is characterized in that, the venom that described step (2) is gathered in the crops, every 1ml is containing virus>=10
8tCID
50;
11. utilize DF1 passage cell to produce the method for pseudorabies living vaccines as claimed in claim 1, it is characterized in that, the pseudorabies DF1 cell live vaccine that described step (3) obtains, and every part vaccine is containing virus>=10
6tCID
50.
12. 1 kinds of pseudorabies living vaccines, is characterized in that, described pseudorabies living vaccines is obtained by the arbitrary described method of claim 1-11.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410515820.4A CN105521487A (en) | 2014-09-30 | 2014-09-30 | Method for preparing pseudorabies live vaccines from DF1 continuous cells and product prepared by method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410515820.4A CN105521487A (en) | 2014-09-30 | 2014-09-30 | Method for preparing pseudorabies live vaccines from DF1 continuous cells and product prepared by method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105521487A true CN105521487A (en) | 2016-04-27 |
Family
ID=55764229
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410515820.4A Pending CN105521487A (en) | 2014-09-30 | 2014-09-30 | Method for preparing pseudorabies live vaccines from DF1 continuous cells and product prepared by method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105521487A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108815517A (en) * | 2018-07-13 | 2018-11-16 | 广东永顺生物制药股份有限公司 | A kind of Duck plague live vaccine and preparation method thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101695573A (en) * | 2009-10-26 | 2010-04-21 | 广东永顺生物制药有限公司 | Method for producing pseudorabies living vaccines by using subculture cell source and product thereof |
| CN102988974A (en) * | 2012-12-14 | 2013-03-27 | 山东滨州沃华生物工程有限公司 | Method for preparing porcine pseudorabies live vaccine by using passage CEF (chicken embryo fibroblast) |
-
2014
- 2014-09-30 CN CN201410515820.4A patent/CN105521487A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101695573A (en) * | 2009-10-26 | 2010-04-21 | 广东永顺生物制药有限公司 | Method for producing pseudorabies living vaccines by using subculture cell source and product thereof |
| CN102988974A (en) * | 2012-12-14 | 2013-03-27 | 山东滨州沃华生物工程有限公司 | Method for preparing porcine pseudorabies live vaccine by using passage CEF (chicken embryo fibroblast) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108815517A (en) * | 2018-07-13 | 2018-11-16 | 广东永顺生物制药股份有限公司 | A kind of Duck plague live vaccine and preparation method thereof |
| CN108815517B (en) * | 2018-07-13 | 2021-09-14 | 广东永顺生物制药股份有限公司 | Duck plague live vaccine and preparation method thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110093307A (en) | The method for adapting to the BHK-21-SC cell strain of serum free suspension culture and preparing vaccine antigen with the cell strain | |
| CN103550771B (en) | The production method of transmissible gastro-enteritis virus vaccine | |
| CN109913404A (en) | The preparation method of infections chicken cloacal bursa virus live vaccine | |
| CN102068695B (en) | Method for producing quadruple inactivated vaccine for newcastle disease, infectious bronchitis, avian influenza (H9 subtype) and infectious bursal disease | |
| CN111662870A (en) | Application of BCG polysaccharide nucleic acid in CIK cell in-vitro culture and preparation of tumor medicine | |
| CN104152403B (en) | A kind of method that establishing goose embryonic epithelium cell line and the goose embryonic epithelium cell line of foundation | |
| CN105274064B (en) | A kind of duck tembusu virus attenuated vaccine strain and its application | |
| CN103977400B (en) | Method for producing marek disease live vaccine of chicken by using cell line | |
| CN110643522A (en) | Culture medium, culture method and application of pasteurella multocida | |
| CN108815517A (en) | A kind of Duck plague live vaccine and preparation method thereof | |
| CN105521487A (en) | Method for preparing pseudorabies live vaccines from DF1 continuous cells and product prepared by method | |
| CN106834168A (en) | A kind of streptococcus suis 2-type low virulent strain and its application | |
| CN113957007B (en) | Inactivated vaccine for mycoplasma synoviae | |
| CN102139104A (en) | Production method for triple inactivated vaccine for newcastle disease, avian influenza (H9 subtype) and infectious bursal disease | |
| CN103127496B (en) | Type III herpes turkey virus freeze dried vaccine | |
| CN104388391A (en) | Paneth cell hybridoma cell strain of mice as well as preparation method and application paneth cell hybridoma cell strain | |
| CN105039241A (en) | Pelodiscus sinensis heart cell continuous cell line and establishing method and ultra-low-temperature cryopreservation method thereof | |
| CN106387314A (en) | Applications of Bacteroides fragilis in animal breeding | |
| CN106867973A (en) | A kind of method that pseudorabies disease vaccine is produced using full suspension technology | |
| CN104274829A (en) | Vaccine composition and preparation method and application thereof | |
| CN103861096A (en) | Preparation method of live vaccine for treating porcine reproductive and respiratory syndrome with high pathogenicity and live vaccine product | |
| CN106237324A (en) | A kind of method using full suspension technology to produce transmissible gastroenteritis of swine vaccine | |
| CN103157107A (en) | Chicken Marek's disease vaccine produced by using continuous passage cell line and production method | |
| CN106367399B (en) | A method of pig parvoviral disease vaccine is produced using full suspension technology | |
| CN112481188A (en) | Preparation method of mixed bacterium agent for efficiently producing beta-glucuronidase |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20160427 |
|
| WD01 | Invention patent application deemed withdrawn after publication |