CN104894054A - Monkey embryo renal epithelial cell Marc-145 suspension adapted strain and application thereof in culture of PRRSV (porcine reproductive and respiratory syndrome virus) and production of PRRSV vaccine - Google Patents
Monkey embryo renal epithelial cell Marc-145 suspension adapted strain and application thereof in culture of PRRSV (porcine reproductive and respiratory syndrome virus) and production of PRRSV vaccine Download PDFInfo
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Abstract
The invention relates to a monkey embryo renal epithelial cell Marc-145 suspension adapted strain and an application thereof in the culture of PRRSV (porcine reproductive and respiratory syndrome virus) and production of a PRRSV vaccine, belonging to the field of biotechnology. The monkey embryo renal epithelial cell Marc-145 suspension adapted strain provided by the invention is collected in the China Center for Type Culture Collection, with a collection number CCTCC No:C201542. The invention also discloses a method for culturing PRRSV by use of the Marc-145 cell suspension adapted strain. By adopting the Marc-145 cell suspension adapted strain provided by the invention, full suspension culture of the Marc-145 cell in a culture medium is realized, and thus the PRRSV is produced by use of the Marc-145 cell suspension adapted strain, step-by-step magnification using a bioreactor is easy to realize in industrial production, and large-scale production of PRRSV vaccine is realized; and compared with the existing spinner-bottle and carrier suspension culture technology, the production technology is simplified, the production cycle is shortened, the production cost is reduced, and the quality and yield of PRRSV vaccine are further improved.
Description
Technical field
The present invention relates to a kind of monkey embryo kidney epithelial cell Marc-145 suspension adapted strain and cultivating reproductive and respiratory syndrome virus, the application of producing in blue ear virus vaccines, belong to biological technical field, more particularly, the present invention relates to a kind of method utilizing bio-reactor suspension Marc-145 cells produce reproductive and respiratory syndrome virus entirely.
Background technology
Marc-145 cell and monkey embryo kidney epithelial cell, derive from monkey-kidney cells, is the clone strain of parent cell (MA104 cell), Anchorage Dependent Growth, can cultivate, without tumorigenicity in continuous passage, this cell strain obtains suspension adapted strain through the domestication of suspension for several times, can not rely on carrier suspension growth.This cell is responsive especially to porcine reproductive and respiratory syndrome virus (PRRSV), is widely used in the production of blue-ear disease vaccine at present.
Because of original Marc-145 cell attachment dependency growth, so the production of blue-ear disease vaccine mainly adopts traditional rolling bottle technique at present, each production batch prepared by vaccine antigen needs hundreds of even thousands of rolling bottles, and each rolling bottle is independent cultivation operating unit, in a production cycle each rolling bottle to inoculate through cell, virus inoculation, virus harvest No. three taphole drilling machine meetings, not only the large pollution probability of labour intensity is high for this, and because the quality of every bottle of cell, viral level are not quite similar, make vaccine quality unstable, differences between batches are large; In addition, adopt cell spinner bottle culture process, not only production efficiency is low, also easily pollutes, and virus titer is unstable, and this traditional technology cannot meet large-scale industrial Production requirement, is automated gradually to produce to substitute.
Because of the growth of Marc-145 cell attachment dependency, mainly carrier dependant cell suspension culture is studied in the automatic production of current blue ear vaccine, and the patent that patent publication No. is CN102002482B " pig breathes and the production method of breeding difficulty syndrome virus " is CN102552896B " a kind of method utilizing bio-reactor to prepare porcine reproductive and respiratory syndrome vaccine " with patent publication No. all discloses scraps of paper carrier cell and cultivates the method for producing reproductive and respiratory syndrome virus; Patent CN102038942B " method of applying biological reactor suitability for industrialized production blue-ear disease vaccine " discloses the method utilizing microcarrier cell suspension culture method to produce reproductive and respiratory syndrome virus; Although microcarrier, scraps of paper carrier cultivation level of automation is high; but cell cultures in essence or adherent culture; carrier cost is relatively high; great majority are single use; and Growth of Cells can not maximize growth by many factors restrictions such as carriers; cell density increase rate is limited, and especially in industrial production amplification, the general volume of culture of carrier suspension culture is all below 300 liters, and this birth defect limits the large-scale application of this technology.
Summary of the invention
The present invention is the defect solving above-mentioned existing culture process, provide a kind of production cost low, can mass-producing amplification culture monkey embryo kidney epithelial cell Marc-145 suspension adapted strain and cultivation reproductive and respiratory syndrome virus, the application of producing in blue ear virus vaccines.
The present invention solves problems of the prior art by the following technical solutions:
A kind of monkey embryo kidney epithelial cell Marc-145 suspension adapted strain, called after Marc-145S, be deposited in China typical culture collection center on April 22nd, 2015, deposit number is CCTCC NO:C201542.
Monkey embryo kidney epithelial cell Marc-145 suspension adapted strain is cultivating the application in reproductive and respiratory syndrome virus.
Monkey embryo kidney epithelial cell Marc-145 suspension adapted strain is producing the application in blue-ear disease vaccine.
Monkey embryo kidney epithelial cell Marc-145 suspension adapted strain cultivates a method for reproductive and respiratory syndrome virus, comprises the steps:
(1) by monkey embryo kidney epithelial cell Marc-145 suspension adapted strain Secondary Culture according to claim 1, be then inoculated into suspension culture in bio-reactor, obtain Marc-145 cell suspending culture solution;
(2) when cell density in Marc-145 cell suspending culture solution reaches 2-5 × 10
6individual/ml time, inoculation reproductive and respiratory syndrome virus;
(3) continue to cultivate reproductive and respiratory syndrome virus, when viable cell density is lower than 0.5 × 10
6individual/ml time results virus liquid.
Obtain Marc-145 cell suspending culture solution in step (1) to comprise the following steps:
1. get the monkey embryo kidney epithelial cell Marc-145 suspension adapted strain of liquid nitrogen cryopreservation, add after fast melt and fill in the shaking flask of nutritive medium, go down to posterity amplification culture;
2. by step 1. in the Marc-145 cell suspension adapted strain of amplification culture be diluted to 0.5 × 10
6the density of individual/ml, inoculates into bio-reactor, expects that blue staining detects cell density and vigor with platform.
The blue otopathy poison of inoculation in step (2): in Marc-145 cell suspending culture solution, cell density is greater than 3.0 × 10
6individual/ml, when cell viability is greater than 90%, utilizes revolving filter entrapped cell, change the nutritive medium of 20-50%, final is the nutritive medium containing 0.5 ~ 2.0% new-born calf serum, and inoculated by reproductive and respiratory syndrome virus in bio-reactor, dosage of inoculation is 1.0 ~ 5.0% of substratum final volume.
Described reproductive and respiratory syndrome virus is pig blue-ear disease living vaccine or inactivated vaccine kind poison, virus titer>=10
7.0tCID
50/ ml.
Described bio-reactor is industrialization stirring type bioreactor.
Compared with prior art; Marc-145 suspension adapted strain production blue-ear disease vaccine method level of automation disclosed by the invention is high; carrier is not needed to get involved; go down to posterity without the need to cell tryptase enzymic digestion, easy mass-producing is amplified, and the present invention simplifies the production technique of blue-ear disease vaccine; reduce its production cost; improve the quality of vaccine, solve differences between batches problem, have unrivaled industrial advantages.
Accompanying drawing explanation
Monkey embryo kidney epithelial cell Marc-145 suspension adapted strain, called after Marc-145S, is deposited in China typical culture collection center, and preservation date is on April 22nd, 2015, and deposit number is CCTCC NO:C201542.
Fig. 1 is Marc-145 cell suspension growth curve chart.
Fig. 2 is that Marc-145 cell inoculates displaing micro picture (100 times of amplifications) before blue otopathy poison.
Fig. 3 is that Marc-145 cell inoculates displaing micro picture (100 times of amplifications) after blue otopathy poison.
Embodiment
Be below specific embodiment of the present invention, to set forth the present invention further, but can not be construed as limiting the scope of the invention.Art technology people it should be understood that and can modify to the details of technical solution of the present invention and form or replace down without departing from the spirit and scope of the present invention, but these amendments or replacement all fall within the scope of protection of the present invention.
The experimental technique used in following embodiment if no special instructions, is ordinary method.
test the primary biological material and facility related to
Monkey embryo kidney epithelial cell Marc-145 suspension adapted strain, be deposited in China typical culture collection center, preservation address is Wuhan City, Hubei Province Wuhan University, and preservation date is on April 22nd, 2015, and deposit number is CCTCC NO:C201542.
Cell growth medium: GRH company M3 special culture media, adds 3% new-born calf serum, and namely cell growth medium is Marc-145 cell suspension strain shake-flask culture and the full suspension culture of bio-reactor nutritive medium used;
Virus culture maintain base: GRH company M3 special culture media, adds 1% new-born calf serum, and namely virus culture maintain base is the Marc-145 cell cultures blue otopathy poison of full suspension culture nutritive medium used;
Reproductive and respiratory syndrome virus: reproductive and respiratory syndrome virus is pig blue-ear disease living vaccine or inactivated vaccine kind poison, and virus titer is more than or equal to 10
7.0tCID
50/ ml, selects TJM-92 strain in this experiment, for commercial goods living vaccine is separated;
Bio-reactor: U.S. NBS 14 liters of bio-reactors.
embodiment 1monkey embryo kidney epithelial cell Marc-145 suspension adapted strain amplification cultivation
China typical culture collection center will be deposited in, deposit number is the monkey embryo kidney epithelial cell Marc-145 suspension adapted strain Secondary Culture of CCTCC NO:C201542, then be inoculated into suspension culture in bio-reactor, obtain Marc-145 cell suspending culture solution, concrete steps are as follows:
1. get the monkey embryo kidney epithelial cell Marc-145 suspension adapted strain of liquid nitrogen cryopreservation, add after fast melt and fill in the shaking flask of nutritive medium, cultivate 60 ~ 72h in 36 ~ 37 DEG C, to go down to posterity amplification culture according to the ratio of 1:3 ~ 1:5;
2. by step 1. in the Marc-145 cell suspension adapted strain of amplification culture be diluted to 0.5 × 10
6the density of individual/ml, inoculates into bio-reactor, and culture temperature is 36 ~ 37 DEG C, and pH value is 6.8 ~ 7.2, and dissolved oxygen is 40% ~ 70%, and cell growth curve as shown in Figure 1;
3. the mensuration of cell density in bio-reactor: with bio-reactor cultured continuously three batches of Marc-145 cells, the sampling in the 72nd hour of cultivating, with trysinization after cell centrifugation, expect that blue staining detects cell density with platform, experimental result is as shown in table 1.
table 1
embodiment 2the titre that Different vaccine dosage cultivates reproductive and respiratory syndrome virus compares
With 5 500ml shake-flask culture Marc-145 suspension cells, inoculating cell is 0.58 × 10
6individual/ml, cultivate 72 hours under same culture conditions, cell density all increases to and is greater than 3 × 10
6individual/ml time, according to 1.0%, 2.0%, 3.0%, 4.0%, the 5.0% inoculation blue otopathy poison TJM-92 strain of substratum final volume, viable cell density is lower than 0.5 × 10
6individual/ml time results virus.Conventionally measure virus titer (TCID
50/ ml), the results are shown in Table 2.
table 2
embodiment 3different training methods cultivate the comparison of reproductive and respiratory syndrome virus titre
Select well-grown adherent Marc-145 cell 2 rolling bottles, select 2 500ml shake-flask culture Marc-145 suspension cells, treat that rolling bottle cell covers with individual layer, shaking flask cell density is greater than 3 × 10
6after individual/ml, according to the 3.0% inoculation blue otopathy poison TJM-92 strain of substratum final volume, when rolling bottle cell CPE reaches 70%, shaking flask cell density is less than 0.5 × 10
6individual/ml time gather in the crops virus liquid respectively, after freeze thawing 2 times, measure virus titer (TCID
50/ ml), the results are shown in Table shown in 3.
table 3
embodiment 4full suspension Marc-145 cell cultures reproductive and respiratory syndrome virus
When the Marc-145 cell density of suspension culture in bio-reactor increases 2-5 × 10
6individual/ml time, inoculation reproductive and respiratory syndrome virus, concrete steps are: in Marc-145 cell suspending culture solution, cell density is greater than 3.0 × 10
6individual/ml, when cell viability is greater than 90%, utilize revolving filter entrapped cell, change the nutritive medium of 20-50% in reactor, final is the nutritive medium containing 0.5 ~ 2.0% new-born calf serum, and enter in bio-reactor by blue ear virus inoculation, dosage of inoculation is 1.0 ~ 5.0% of substratum final volume, described blue otopathy poison is pig blue-ear disease living vaccine or inactivated vaccine kind poison, virus titer>=10
7.0tCID
50/ ml.At 35 DEG C ~ 37 DEG C, pH value is 6.8 ~ 7.2, and dissolved oxygen is 40% ~ 70% cultivation reproductive and respiratory syndrome virus 36 ~ 72h, and viable cell density is lower than 0.5 × 10
6individual/ml time results virus.Marc-145 cell inoculates displaing micro picture before and after blue otopathy poison as shown in Figure 2 and Figure 3, and as can be seen from the figure connecing the cell density after poison, to reduce amplitude large, and cytopathy is obvious; The cultivation titre of 3 batches is in table 4.
table 4
embodiment 5the preparation of blue-ear disease vaccine
According to embodiment 4 continuous production 3 batches of reproductive and respiratory syndrome virus liquid, results virus liquid, as antigen for vaccine liquid, measures viral level (TCID
50/ ml); reference " high-pathogenicity porcine reproductive and respiration syndrome living vaccine (TJM-F92 strain) " quality standard of simultaneously sampling carries out the inspection of semifinished product; dilute by a certain percentage after qualified and add 20% heat-resisting lyophilized protecting agent, quantitative separating after mixing, carries out vacuum freezedrying in Freeze Drying Equipment.Vaccine finished product is tested with reference to China's existing " high-pathogenicity porcine reproductive and respiration syndrome living vaccine (TJM-F92 strain) " quality standard, and indices is all qualified.
Claims (8)
1. a monkey embryo kidney epithelial cell Marc-145 suspension adapted strain, is characterized in that: called after Marc-145S, and be deposited in China typical culture collection center on April 22nd, 2015, deposit number is CCTCC NO:C201542.
2. monkey embryo kidney epithelial cell Marc-145 suspension adapted strain according to claim 1 is cultivating the application in reproductive and respiratory syndrome virus.
3. monkey embryo kidney epithelial cell Marc-145 suspension adapted strain according to claim 1 is producing the application in blue-ear disease vaccine.
4. monkey embryo kidney epithelial cell Marc-145 suspension adapted strain cultivates a method for reproductive and respiratory syndrome virus, it is characterized in that, comprises the steps:
By monkey embryo kidney epithelial cell Marc-145 suspension adapted strain Secondary Culture according to claim 1, be then inoculated into suspension culture in bio-reactor, obtain Marc-145 cell suspending culture solution;
When in Marc-145 cell suspending culture solution, cell density reaches 2-5 × 10
6individual/ml time, inoculation reproductive and respiratory syndrome virus;
Continue to cultivate reproductive and respiratory syndrome virus, when viable cell density is lower than 0.5 × 10
6individual/ml time results virus liquid.
5. a kind of monkey embryo kidney epithelial cell Marc-145 suspension adapted strain according to claim 4 cultivates the method for reproductive and respiratory syndrome virus, it is characterized in that, obtains Marc-145 cell suspending culture solution and comprise the following steps in step (1):
1. get the monkey embryo kidney epithelial cell Marc-145 suspension adapted strain of liquid nitrogen cryopreservation, add after fast melt and fill in the shaking flask of nutritive medium, go down to posterity amplification culture;
2. by step 1. in the Marc-145 cell suspension adapted strain of amplification culture be diluted to 0.5 × 10
6the density of individual/ml, inoculates into bio-reactor, expects that blue staining detects cell density and vigor with platform.
6. a kind of monkey embryo kidney epithelial cell Marc-145 suspension adapted strain according to claim 4 cultivates the method for reproductive and respiratory syndrome virus, it is characterized in that: the blue otopathy poison of inoculation in step (2): in Marc-145 cell suspending culture solution, cell density is greater than 3.0 × 10
6individual/ml, when cell viability is greater than 90%, utilizes revolving filter entrapped cell, change the nutritive medium of 20-50%, final is the nutritive medium containing 0.5 ~ 2.0% new-born calf serum, and inoculated by reproductive and respiratory syndrome virus in bio-reactor, dosage of inoculation is 1.0 ~ 5.0% of substratum final volume.
7. a kind of monkey embryo kidney epithelial cell Marc-145 suspension adapted strain according to claim 4 cultivates the method for reproductive and respiratory syndrome virus, it is characterized in that: described reproductive and respiratory syndrome virus is pig blue-ear disease living vaccine or inactivated vaccine kind poison, virus titer>=10
7.0tCID
50/ ml.
8. a kind of monkey embryo kidney epithelial cell Marc-145 suspension adapted strain according to claim 4 cultivates the method for reproductive and respiratory syndrome virus, it is characterized in that: described bio-reactor is industrialization stirring type bioreactor.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105950544A (en) * | 2016-05-17 | 2016-09-21 | 西北民族大学 | Domestication method of full suspension culture type Marc-145 cell line |
| CN106237323A (en) * | 2016-09-22 | 2016-12-21 | 齐鲁动物保健品有限公司 | Pig blue-ear disease purified vaccine and preparation method thereof |
| CN110241089A (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 reproductive and respiratory syndrome virus antigen |
| CN110373379A (en) * | 2019-07-25 | 2019-10-25 | 北京鼎持生物技术有限公司 | A kind of method that the full suspension cell of Marc-145 directly cultivates PRRS virus vaccine |
Citations (1)
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| CN102453699A (en) * | 2010-10-18 | 2012-05-16 | 北京清大天一科技有限公司 | Sensitive cell suspension culture method and method for producing blue-ear disease vaccine by using sensitive cell suspension culture method |
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Patent Citations (1)
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| CN102453699A (en) * | 2010-10-18 | 2012-05-16 | 北京清大天一科技有限公司 | Sensitive cell suspension culture method and method for producing blue-ear disease vaccine by using sensitive cell suspension culture method |
Non-Patent Citations (2)
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| 冯磊 等: ""Marc － 145 细胞无血清培养驯化、代谢分析及PRRSV 增殖能力比较"", 《江苏农业科学》 * |
| 陈文庆 等: ""悬浮培养工艺与转瓶培养工艺的比较分析"", 《中国兽药杂志》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105950544A (en) * | 2016-05-17 | 2016-09-21 | 西北民族大学 | Domestication method of full suspension culture type Marc-145 cell line |
| CN105950544B (en) * | 2016-05-17 | 2020-07-03 | 西北民族大学 | Domestication method of full suspension culture type Marc-145 cell line |
| CN106237323A (en) * | 2016-09-22 | 2016-12-21 | 齐鲁动物保健品有限公司 | Pig blue-ear disease purified vaccine and preparation method thereof |
| CN106237323B (en) * | 2016-09-22 | 2019-12-06 | 齐鲁动物保健品有限公司 | Porcine reproductive and respiratory syndrome purified vaccine and preparation method thereof |
| CN110241089A (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 reproductive and respiratory syndrome virus antigen |
| CN110373379A (en) * | 2019-07-25 | 2019-10-25 | 北京鼎持生物技术有限公司 | A kind of method that the full suspension cell of Marc-145 directly cultivates PRRS virus vaccine |
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