CN103157107A - Chicken Marek's disease vaccine produced by using continuous passage cell line and production method - Google Patents
Chicken Marek's disease vaccine produced by using continuous passage cell line and production method Download PDFInfo
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- CN103157107A CN103157107A CN2011104201400A CN201110420140A CN103157107A CN 103157107 A CN103157107 A CN 103157107A CN 2011104201400 A CN2011104201400 A CN 2011104201400A CN 201110420140 A CN201110420140 A CN 201110420140A CN 103157107 A CN103157107 A CN 103157107A
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Abstract
The invention discloses a production method for a chicken Marek's disease vaccine by using a continuous passage cell line. The method comprises the following steps: (1) passage and culture of a cell used for vaccine preparation; (2) propagation of a cellular virus seed; (3) inoculation of a virus; (4) centrifugal extraction of the cell for collection of the virus; and (5) distribution and split charging of the vaccine. According to the invention, passage cells are used to replace original primary chicken embryonic fibroblasts for production of the chicken Marek's disease vaccine, so production cost is substantially reduced; the production method has a simple process, is stable and is easy to operate; virus plaque content is high, the vaccines of different batches have small difference, vaccine quality is easily controllable, and output and quality of the vaccine can be substantially improved. The chicken Marek's disease vaccine produced in the invention has high immune efficacy on a standard virulent strain and has an immune effect superior to that of a product of a same kind.
Description
Technical field
The present invention relates to a kind ofly produce chicken Marek's disease vaccine and method with continuous cell line, refer to especially a kind ofly produce chicken Marek's disease vaccine and method thereof with Carnis Coturnicis japonicae sarcoma fibrocyte QT6.
Background technology
Marek (Marek ' s disease, MD) be by the marek's disease virus of herpetoviridae (Marek ' s disease virus, MDV) a kind of infectiousness neoplastic disease of the chicken that causes, form feature with lymphadenosis and tumor, at peripheral nervous, gonad, the various internal organs of iris, muscle and skin generation monocyte infiltration.When MD causes tumor and death clinically, the more important thing is the immune organ of infringement infected chicken, cause immunosuppressant, cause Abwehrkraft des Koepers to descend and vaccinated immunne response is reduced or do not reply, cause the concurrent and secondary infection of other various diseases, cause huge economic loss.
Vaccination is still one of effective way of present control Marek.The commercialization chicken Marek's disease vaccine that uses at present is mostly as the seedling material with primary chick embryo fibroblast.The method for preparing primary cell is not only time-consuming but also cost is high.About 30% the cost of producing vaccine is for the preparation of chick embryo fibroblast.Production of vaccine extremely relies on the supply of SPF hatching egg.SPF fowl group raises under specific condition and proves that regularly it does not contain avian pathogens.Any interruption of the supply of SPF hatching egg all will be interrupted the production of MDV vaccine.And the primary cell labor intensity of each preparation is large, and differences between batches are also large, thereby cause that quality is unstable, and operating process is easily polluted, and vaccine output and quality are extremely restricted.
Poultry farming industry is badly in need of can be used for producing the continuous cell line of Mareks disease vaccine all the time.Although developed many birdss or mammal cell line (as EP0748867 and EP0884382), still do not have continuous cell line can be in Mareks disease vaccine as the effective substitute of chick embryo fibroblast (CEF).
Summary of the invention
In view of this, but main purpose of the present invention is to provide a kind of continuous cell line of utilization continuous passage to produce Mareks disease vaccine, with realize by uses the cultivation of can going down to posterity fibroblast---Carnis Coturnicis japonicae sarcoma fibrocyte is QT6 (preserving number is as ATCC CRL-1708), substitute primary chick embryo fibroblast, be used for serialization commercial production chicken Marek's disease vaccine, thereby obtain a kind of vaccine quality and output is high, batch difference is little, immune efficacy is stable Mareks disease vaccine.
Technical scheme
The Mareks disease vaccine that a kind of continuous cell line is produced wherein, comprises following component in every Mareks disease vaccine finished product (2ml volume):
Ham ' s F12K culture fluid: 1.4～1.7ml;
Hyclone: 0.2～0.4ml;
DMSO： 0.1～0.2ml；
Wherein, marek's disease virus content 〉=6 * 10
6PFU, described marek's disease virus is for using continuous cell line production to obtain.
Preferably, passage cell of the present invention is that quail meat tumor fibrocyte is QT6, and this cell is the fibroblast that can go down to posterity and cultivate, available from ATCC CRL-1708.
Preferably, marek's disease virus of the present invention is marek's disease virus CVI988/Rispens strain, be commercial Strain, and open in " research of chicken Marek's disease CVI988_Rispens strain biological characteristics " (Chinese veterinary's magazine, the 38th the 5th phase of volume in 2002).
The present invention is not particularly limited the type of marek's disease virus, and preferably, marek's disease virus of the present invention is any one or a plurality of combination in serum I type, serum II type or serum II I type.
Preferably, Mareks disease vaccine of the present invention is liquid nitrogen vaccine or freeze dried vaccine.
Above-mentioned marek's disease virus and Mareks disease vaccine obtain by following method, namely produce the method for Mareks disease vaccine with continuous cell line, comprise the following steps:
1) the going down to posterity and cultivating of passage cell: use the cell dispersion liquid to carry out had digestive transfer culture passage cell, cultivate with cell growth medium, to form good cell monolayer;
2) breeding of cell seed culture of viruses: with marek's disease virus in step 1) the passage cell subculture of the good cell monolayer of the formation that obtains, to obtain work kind poison;
The passage cell of the cell monolayer that the formation that the 3) breeding of work kind of poison: with step 1) obtains is good discards cell growth medium, and inoculation contains the work kind poison of cell maintenance medium, cultivates under 37～38 ℃;
4) when being cultured to CPE 〉=80% when above, the results marek's disease virus, and join the Seedling packing to obtain Mareks disease vaccine.
Wherein, joining the method for gathering in the crops marek's disease virus before Seedling is:
When being cultured to the pathogenic change effect of cell appearance more than 80%, can gather in the crops, discard cell maintenance medium, add EDTA-pancreatin cell dispersion liquid, after making Digestive system and cell fully contacting, discard trypsin solution, add cell maintenance medium, stop digestion, the results marek's disease virus, after steriling test, divide to centrifuge bottle; In 2～8 ℃, the centrifugal 10～20min of 1500r/min, centrifugal rear supernatant discarded, collecting precipitation cell (it contains marek's disease virus).
Culture fluid and the ratio used when wherein, joining Seedling are as follows:
Ham ' s F12K the culture fluid (not increase serum) of volume 75～85%, pH value is adjusted to 7.2; The DMSO of the hyclone of volume 10～20% and volume 5～10% adds in the sedimentation cell of collection, shakes up, and the steriling test before results is done in sampling, and after carrying out viable count, quantitative separating seals in aseptic ampoule bottle.
Preferably, step 4 in said method of the present invention) also comprise the Mareks disease vaccine that obtains is lowered the temperature, during to-90 ℃～-120 ℃, vaccine is put into liquid nitrogen and preserve, to obtain the process of Marek liquid nitrogen vaccine.
Preferably, in said method of the present invention, passage cell is that Carnis Coturnicis japonicae sarcoma fibrocyte is QT6.
Preferably, marek's disease virus described in said method of the present invention is marek's disease virus CVI988/Rispens strain.
Preferably, in said method of the present invention, marek's disease virus is any one or a plurality of combination in serum I type, serum II type or serum II I type.
Technique effect
The present invention uses passage cell to produce the method for chicken Marek's disease vaccine, has the following advantages at least:
1) need not all to prepare primary chick embryo fibroblast when each production vaccine, not only greatly reduce production cost, also simplified operation sequence, production technology simple and stable, easy to operate significantly improves viral yield.
2) the chook MDV vaccine quality of the present invention's production is good, and differences between batches are little, and immune efficacy is high, and immune effect is stable.
Description of drawings
Fig. 1 is that the present invention uses passage cell to produce the flow chart of chicken Marek's disease vaccine.
The specific embodiment
The invention is further described below in conjunction with Figure of description and specific embodiment:
The method that embodiment 1 passage cell is produced the chicken Marek's disease vaccine
(1) seedling going down to posterity and cultivating with the QT6 cell:
The QT6 cell is through EDTA-pancreatin cell dispersion liquid had digestive transfer culture, and one bottle passes three bottles, continues to cultivate every 4 days biography generation in 37 ℃ with cell growth medium.When forming good monolayer, namely can be used for continuing to go down to posterity or virus inoculation.The cell inoculum density is 2 * 10
5Cells/ml;
(2) breeding of cell seed culture of viruses:
Get the chook MDV CVI988/Rispens strain that liquid nitrogen is preserved, 1～2 generation of rejuvenation subculture on above-mentioned cell, preparation work kind poison;
(3) inoculation work seed culture of viruses:
Get the 10000ml cell spinner bottle that forms good monolayer, discard cell growth medium, inoculation contains the maintenance medium 1000ml of CVI988/Rispens strain, and every milliliter contains 1～20,000 and contains poison cell, rotation cultivation under 37～38 ℃.
(4) results:
When being cultured to the pathogenic change effect of cell appearance more than 80%, can gather in the crops, discard maintenance medium, add 28ml EDTA-pancreatin cell dispersion liquid, after making Digestive system and cell fully contacting, discard trypsin solution, add maintenance medium, stop digestion, results after steriling test, are divided to centrifuge bottle;
(5) centrifugal extraction cell:
In 2～8 ℃, the centrifugal 10～20min of 1500r/min, centrifugal rear supernatant discarded, collecting precipitation cell;
(6) join the Seedling packing:
To join Seedling volume 75～85% culture fluid, join the hyclone of Seedling volume 10～20% and join the DMSO of Seedling volume 5～10%, add after mixing in the sedimentation cell of collection, shake up, the steriling test before results is done in sampling, after carrying out viable count, divide to install in aseptic ampoule bottle the 2ml/ bottle, sealing makes every milliliter of contained viral plaque number be not less than 3 * 10
6PFU;
(7) programmed cooling:
Utilize the programmed cooling instrument to carry out programmed cooling, vaccine is put into liquid nitrogen until the Seedling temperature drop to-90 ℃～-120 ℃ the time and preserve;
Above-mentioned steps can be with reference to accompanying drawing 1 of the present invention.
(8) vaccine diluent preparation:
Broth medium 50ml; Pidolidone list sodium 0.83g; KH
2PO
40.52g; K
2HPO
43H
2O 1.64g; Sucrose 76.62g adds deionized water and is made into 1000ml, 115 ℃ of autoclaving 20min, through check aseptic after 4 ℃ of preservations, before vaccine uses, dilution is used.
Above-mentioned cell growth medium is the Ham ' s F12K culture fluid of 5% hyclone concentration, and pH value is adjusted to 7.2; Maintenance medium is the Ham ' s F12K culture fluid of 3% hyclone concentration, and pH value is adjusted to 7.4; Join Seedling not increase serum of Ham ' s F12K culture fluid, pH value is adjusted to 7.2.The manufacturer of hyclone is PAA Laboratories GmbH.
The compound method of above-mentioned EDTA-pancreatin cell dispersion liquid:
0.02% disodium ethylene diamine tetra-acetic acid solution preparation (EDTA): disodium ethylene diamine tetra-acetic acid solution preparation (EDTA) 0.2g; NaCl 8.0g; Na
2HPO
412H2O 1.15g; KCl 0.2g; KH
2PO
40.2g; Glucose 0.2g; Mend deionized water to the rear packing of 1000ml dissolving, 4 ℃ of preservations of autoclaving (15 pounds of 15min);
0.25% trypsin: trypsin 0.25g is dissolved in the PBS of 100ml 0.01M PH7.4, filters packing ,-20 ℃ of preservations.
0.01M the PBS:7.9g NaCl of PH7.4,0.2g KCl, 0.24g KH
2PO
4(or 1.44gNa
2HPO
4) and 1.8g K
2HPO
4, be dissolved in the 800ml deionized water, transfer pH value to 7.4, add at last deionized water and be settled to 1000ml.Preserve in 4 ℃ of refrigerators and get final product.
Facing used time 0.25% trypsin mixes by 1: 4 with 0.02% disodium ethylene diamine tetra-acetic acid solution preparation (EDTA) and is EDTA-pancreatin cell dispersion liquid.
Embodiment 2 chicken Marek's disease vaccines and the comparative study of using the CEF cell to prepare the chicken Marek's disease vaccine
The 1 existing chicken Marek's disease vaccine key step of preparation is as follows:
(1) preparation of chick embryo fibroblast (CEF) monolayer:
Select the 10 well-developed SPF Embryo Gallus domesticus of age in days, first with iodine tincture cotton balls sterilization air chamber position, take off iodine with 75% cotton ball soaked in alcohol again, asepticly choose idiosome and be placed in aseptic plate, PBS with 0.1M washs idiosome and removes eyes, pawl and internal organs, then adopt continuous digestion method to prepare the CEF suspension, make the cell density of 1,500,000/ml, inoculating cell culture bottle after mixing.；
(2) breeding of cell seed culture of viruses:
Get the chook MDV CVI988/Rispens strain in embodiment 1,1～2 generation of rejuvenation subculture on above-mentioned CEF, preparation work kind poison;
(3) virus inoculation:
With cultivating the 10000ml rolling bottle of the good cell monolayer of 24h growth conditions, discard culture fluid, inoculation contains the maintenance medium 1000ml of CVI988/Rispens strain, and every milliliter contains 1～20,000 and contains poison cell, rotation cultivation under 37～38 ℃.
(4) results:
When typical cytopathic appears in the cell more than 80%, can gather in the crops.Discard maintenance medium, add 28ml EDTA-pancreatin cell dispersion liquid, after making Digestive system and cell fully contacting, discard trypsin solution, add maintenance medium, stop digestion, results after steriling test, are divided to centrifuge bottle;
(5) centrifugal extraction cell:
In 2～8 ℃, the centrifugal 10～20min of 1500r/min, centrifugal rear supernatant discarded, collecting precipitation cell;
(6) join the Seedling packing:
To join Seedling volume 75～85% culture fluid, join the hyclone of Seedling volume 10～20% and join the DMSO of Seedling volume 5～10%, add after mixing in the sedimentation cell of collection, shake up, the steriling test before results is done in sampling, after carrying out viable count, divide to install in aseptic ampoule bottle the 2ml/ bottle, sealing makes every milliliter of contained viral plaque number be not less than 3 * 10
6PFU;
(7) programmed cooling:
Utilize the programmed cooling instrument to carry out programmed cooling, vaccine is put into liquid nitrogen until the Seedling temperature drop to-90 ℃～-120 ℃ the time and preserve;
Above-mentioned cell growth medium is the M199 culture fluid of 4% hyclone; Maintenance medium is the M199 culture fluid of 2% hyclone; Join Seedling not increase serum of M199 nutritional solution.
(8) vaccine diluent preparation:
Broth medium 50ml; Pidolidone list sodium 0.83g; KH
2PO
40.52g; K
2HPO
43H
2O 1.64g; Sucrose 76.62g adds deionized water and is made into 1000ml, 115 ℃ of autoclaving 20min, through check aseptic after 4 ℃ save backup.
2 immunological comparisons:
With 160 1 age in days SPF chickens that provided by the logical experimental technique company limited of Beijing Cimmeria dimension, be divided at random 4 groups, 40 every group.Totally 2 groups of immune group are inoculated respectively embodiment 1 the chicken Marek's disease vaccine of producing and the chicken Marek's disease vaccine that uses primary chick embryo fibroblast to produce, with every nape subcutaneous injection 0.2ml of section (containing 2000PFU) after the vaccine diluent dilution.Other 2 groups is nonimmune group, and wherein one group is not immune also counteracting toxic substances matched group (blank) not, and another group is counteracting toxic substances matched group (positive controls).4 groups of chickens are raised in isolator respectively, feed feedstuff and drinking-water to sterilization.
3 counteracting toxic substances:
Rear 21 days of immunity is inoculated respectively Marek standard virulent strain capital-1 strain (available from China Veterinery Drug Inspection Office, preserving number CVCC AV86) with immune group and positive controls, and 1ml/ props up.During counteracting toxic substances, make 10 times of dilutions, every chicken lumbar injection 0.5ml with the M199 culture fluid respectively.Continuation is wherein raised in isolation, observes every day, records morbidity and death condition, and dead chicken is in time cutd open inspection, observes pathological changes.After counteracting toxic substances 63 days, the survival chicken is cutd open inspection observe the naked eyes pathological changes, calculate protective index.
4 experimental results:
The counteracting toxic substances protection result of vaccine immunity group:
Result shows, two groups of chicken Marek's disease vaccines all are highly resistant to the attack of the strong poison of MD standard, and protective index is respectively 90.6%, 87.5%.Concrete data see Table 1.
Protective index and death condition table after table 1 immune group and the strong poison of matched group inoculation
Annotate: protective index %=(positive controls MD positive rate-immune group MD positive rate/positive controls MD positive rate) * 100%
Experimental result shows the chook MDV CVI988/Rispens strain live vaccine of producing with passage cell, and superior in quality, capital-1 strain has higher immune efficacy to standard virulent strain, and immune effect is higher than the same based article of primary CEF preparation.
The advantages the such as in addition vaccine produced of the present invention also has that PFU content is high, cost is low, effect stability, operating procedure are simple and easy to control.
The above is only preferred embodiment of the present invention, and is in order to limit the present invention, within the spirit and principles in the present invention not all, any modification of doing, is equal to replacement, improvement etc., within all should being included in protection scope of the present invention.
Claims (10)
1. the Mareks disease vaccine that continuous cell line is produced, is characterized in that,
Every 2ml Mareks disease vaccine finished product comprises following component:
Ham ' s F12K culture fluid: 1.4～1.7ml;
Hyclone: 0.2～0.4ml;
DMSO： 0.1～0.2ml；
Marek's disease virus content 〉=6 * 10
6PFU, wherein, described marek's disease virus is for using continuous cell line production to obtain.
2. Mareks disease vaccine according to claim 1, is characterized in that, described passage cell is that quail meat tumor fibrocyte is QT6.
3. Mareks disease vaccine according to claim 1, is characterized in that, described marek's disease virus is marek's disease virus CVI988/Rispens strain.
4. Mareks disease vaccine according to claim 1, is characterized in that, described marek's disease virus is any one or a plurality of combination in serum I type, serum II type or serum II I type.
5. Mareks disease vaccine according to claim 1, is characterized in that, described Mareks disease vaccine is liquid nitrogen vaccine or freeze dried vaccine.
6. a method of producing Mareks disease vaccine with continuous cell line, is characterized in that, comprises the following steps:
1) passage cell is used the cell dispersion liquid carry out had digestive transfer culture, cultivate with cell growth medium, to form good cell monolayer;
2) with marek's disease virus in step 1) the passage cell subculture of the good cell monolayer of the formation that obtains, to obtain work kind poison;
3) with step 1) passage cell of the good cell monolayer of the formation that obtains, discard cell growth medium, inoculation contains the work kind poison of cell maintenance medium, cultivates under 37～38 ℃, with the breeding marek's disease virus;
4) when being cultured to CPE 〉=80% when above, sedimentation cell, the results marek's disease virus, and join the Seedling packing to obtain Mareks disease vaccine.
7. method according to claim 6, is characterized in that, described step 4) also comprise the Mareks disease vaccine that obtains is lowered the temperature, during to-90 ℃～-120 ℃, vaccine is put into liquid nitrogen and preserve, to obtain the process of Marek liquid nitrogen vaccine.
8. according to claim 6 or 7 described methods, is characterized in that, described passage cell is that Carnis Coturnicis japonicae sarcoma fibrocyte is QT6.
9. according to claim 6 or 7 described methods, is characterized in that, described marek's disease virus is marek's disease virus CVI988/Rispens strain.
10. according to claim 6 or 7 described methods, is characterized in that, described marek's disease virus is any one or a plurality of combination in serum I type, serum II type or serum II I type.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103977400A (en) * | 2014-05-29 | 2014-08-13 | 南京创启生物科技有限公司 | Method for producing marek disease live vaccine of chicken by using cell line |
| CN105408471A (en) * | 2013-07-25 | 2016-03-16 | 国家农艺研究院 | Method for selecting a permissive cell line for replicating avian viruses |
| CN113846067A (en) * | 2021-10-26 | 2021-12-28 | 乾元浩生物股份有限公司 | Method for producing chicken Marek's virus strain and application thereof |
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| CN1256310A (en) * | 1998-12-09 | 2000-06-14 | 辉瑞产品公司 | Process for preparing fowl paralysis virus using continuous avian cell line |
| CN101668539A (en) * | 2007-04-24 | 2010-03-10 | 维涡里斯公司 | Duck embryonic derived stem cell lines for the production of viral vaccines |
| CN102260650A (en) * | 2011-07-13 | 2011-11-30 | 普莱柯生物工程股份有限公司 | Large-scale production method of fowl Marek's disease virus |
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2011
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Patent Citations (3)
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| CN1256310A (en) * | 1998-12-09 | 2000-06-14 | 辉瑞产品公司 | Process for preparing fowl paralysis virus using continuous avian cell line |
| CN101668539A (en) * | 2007-04-24 | 2010-03-10 | 维涡里斯公司 | Duck embryonic derived stem cell lines for the production of viral vaccines |
| CN102260650A (en) * | 2011-07-13 | 2011-11-30 | 普莱柯生物工程股份有限公司 | Large-scale production method of fowl Marek's disease virus |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105408471A (en) * | 2013-07-25 | 2016-03-16 | 国家农艺研究院 | Method for selecting a permissive cell line for replicating avian viruses |
| CN105408471B (en) * | 2013-07-25 | 2019-01-18 | 国家农艺研究院 | Method for screening the cell line to duplication avian viruses receiving |
| CN103977400A (en) * | 2014-05-29 | 2014-08-13 | 南京创启生物科技有限公司 | Method for producing marek disease live vaccine of chicken by using cell line |
| CN103977400B (en) * | 2014-05-29 | 2015-06-17 | 南京创启生物科技有限公司 | Method for producing marek disease live vaccine of chicken by using cell line |
| CN113846067A (en) * | 2021-10-26 | 2021-12-28 | 乾元浩生物股份有限公司 | Method for producing chicken Marek's virus strain and application thereof |
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Application publication date: 20130619 |