CN107988143A - One plant of BHK-21 cells Gs cell line - Google Patents
One plant of BHK-21 cells Gs cell line Download PDFInfo
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- 241000700605 Viruses Species 0.000 claims abstract description 26
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- 238000004114 suspension culture Methods 0.000 claims description 10
- 241000710198 Foot-and-mouth disease virus Species 0.000 claims description 9
- 239000012930 cell culture fluid Substances 0.000 claims description 9
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
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- C12N2770/00011—Details
- C12N2770/32011—Picornaviridae
- C12N2770/32111—Aphthovirus, e.g. footandmouth disease virus
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Abstract
The present invention relates to microorganism field, there is provided one plant of 21 cell line Gs clone strain of BHK, deposit number are：14735.Cell clone condition of culture according to the invention is cultivated, with 0.6 × 106/ml‑1.0×106When/ml is passed on, 48 hour cell density of culture reach 8.0 × 106/ml‑1.2×107/ ml, cell viability is more than 91%；Cultivate 72 hour cell density and reach 1.5 × 107/ml‑2.0×107/ ml, cell viability is more than 92%；Cultivate 96 hour cell vigor and be still in more than 80%.21 cell line Gs clone strains of BHK provided by the present invention can be used for the extensive culture that suspends of animal virus, production of vaccine technique, preferable the advantages that maintaining viral antigen to stablize can be simplified, and the production of vaccine cycle can be shortened, production efficiency is improved, possesses the ability that vaccine supply is emergent in SARS Epidemic.
Description
Technical field
The present invention relates to microorganism field, specifically, is related to one plant of BHK-21 cells Gs cell line.
Background technology
BHK-21 cells, i.e. Syria's baby hamster kidney cell or golden yellow baby hamster kidney cell (Baby Hamster
Kidney cell, BHK-21), the baby hamster kidney from 51 ages in days, the cell is in upper world's sixties by Macpherson
IA, Stoker MGP formally build strain, its initial cell is adherent fibroblast, can continuous passage, be diploid cell, dye
Colour solid is 42.Because of its convenient sources, reproduction speed is fast, and quality and exogenous factor are easy to control, is widely used in the propagation of virus,
It is suitable for the large-scale industrial production of a variety of live vaccines, is more used for the protein product for producing some other high added values.
The cell is domesticated for the stable suspended culture cell of growth by nineteen sixty-five Capstick etc., and first using the training that suspends
Support BHK-21 cells aftosa vaccine is produced in bioreactor, from this open BHK-21 cell suspension cultures it is new when
Generation, hereafter the suspension culture techniques of BHK-21 cells be widely popularized and used in various fields.In China, Xue Ying etc. is realized
The suspension culture of BHK-21 cells, well connect the suspension culture that Na etc. realizes the serum-free of the cell, which exists today
Domestic Duo Jia live vaccines enterprise is still using.But exploitation of the China to BHK-21 cell lines still has necessarily with developed country
Gap, without High Density Cultivation have concurrently high yield poison characteristic cell line be restrict China's animal vaccine industry improve and production
One of key factor of product skill upgrading.
The content of the invention
In order to solve problem of the prior art, the object of the present invention is to provide one plant can High Density Cultivation, and there is high yield
The BHK-21 cells Gs cell lines of malicious characteristic.
Technical scheme is as follows：
In a first aspect, the present invention is based on life CD21 Screening of Media, there is provided one plant of BHK-21 cell line Gs cell line,
Syria hamster nephrocyte BHK-21 clone strains are identified as, entitled BHK-21-Gs, is preserved in Chinese microorganism strain preservation
Administration committee's common micro-organisms center (abbreviation CGMCC, address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Chinese science
Institute of microbiology of institute, postcode：100101), preservation date is on November 10th, 2017, deposit number CGMCC
No.14735。
In order to verify the High Density Cultivation effect of cell line provided by the present invention, the present invention has carried out different cultures first
Temporal Apoptosis situation and vitality test.Research is found, the BHK-21 cells Gs clone strains are seeded to cell
Nutrient solution, carries out cell suspension cultures in bioreactor, and condition of culture is：37 DEG C, pH7.0-7.2, dissolved oxygen amount 65%,
Rotating speed 60-120r/min, cultivates 48-60h；Afterwards with 0.6 × 106/ml-1.0×106When/ml is passed on, culture 48 is thin when small
Born of the same parents' density reaches 8.0 × 106/ml-1.2×107/ml；For cell viability more than 91%, 72 hour cell density of culture reach 1.5
×107/ml-2.0×107/ ml, cell viability is more than 92%；Cultivate 96 hour cell vigor and be still in more than 80%.
Wherein, the cell culture fluid is the CD21 fluid nutrient mediums of pH 7.0-7.2.
In the embodiment of the present invention, used cell culture fluid (is purchased from for GE Life CD21 culture mediums
Invitrogen (Shanghai) Trading Co., Ltd. goods number A16277-03, pH 7.0-7.2)；Used bioreactor is&CELLIGEN 310 Fermentor/Bioreactor。
Further, in order to verify whether the cell line has a high yield poison characteristic, the present invention with pseudorabies virus and
Foot and mouth disease virus distinguishes vaccination target cell, and the cell suspension cultures of BHK-21-Gs cell lines are carried out in bioreactor,
Virus liquid is harvested, has carried out viral quality evaluation.
The present invention is Strain Ea carrying out the pseudorabies virus used in viral quality evaluation, used aftosa
Virus is OS99 plants.
In preceding method, treat in cell culture fluid bhk cell density up to 7.0 × 106/ml-1.0×107It is inoculated with after/ml
Virus, it is 1 that pseudorabies virus is pressed the ratio between virus and total number of cells:1000-1:100 ratio is inoculated with.
And the poison amount suggestion that connects of foot and mouth disease virus is 1000TCID50, therefore when being inoculated with foot and mouth disease virus, treat cell culture
Bhk cell density is up to 7.0 × 10 in liquid6/ml-1.0×107Be inoculated with foot and mouth disease virus after/ml, by foot and mouth disease virus by virus with
The ratio between total number of cells is inoculated with for the ratio of ten a ten thousandths.
After the complete lesion of cell, stop culture, collect supernatant, measure TCID50Value, the results show that 107TCID50/
0.1ml-108TCID50/ 0.1ml scopes are the qualified virus liquid of inspection.
The beneficial effects of the present invention are：
The present invention pass through special screening, obtain one plant can High Density Cultivation, and with high yield poison characteristic BHK-21 it is thin
Born of the same parents system Gs cell lines, available for the extensive culture that suspends of animal virus, can simplify production of vaccine technique, preferable to maintain virus
The advantages that antigen is stablized, and the production of vaccine cycle can be shortened, production efficiency is improved, possesses what vaccine supply in SARS Epidemic was met an urgent need
Ability.
Embodiment
Following embodiments are used to illustrate the present invention, but are not limited to the scope of the present invention.Unless otherwise specified, embodiment
In the conventional means that are well known to those skilled in the art of used technological means, raw materials used is commercial goods.
The percentage sign " % " arrived involved in the present invention, if not specified, refers to mass percent；But the percentage of solution
Than unless otherwise specified, referring to the grams containing solute in 100ml solution；Percentage between liquid, refers to hold at 20 DEG C
The ratio of amount.Unless otherwise noted, heretofore described " part " refers to " parts by weight ".
Cell culture fluid used, viral growth liquid are that GE Life CD21 culture mediums (are purchased from the English Weihe River in following embodiments
Jie Ji (Shanghai) trade Co., Ltd goods number A16277-03, pH 7.0-7.2).
Bioreactor used is&CELLIGEN 310 Fermentor /Bioreactor。
The screening of 1 BHK-21 cells Gs cell lines of embodiment
1.BHK-21 cells pass on：Take the BHK-21 suspended culture cells for cultivating for two generations after recovering, growth 48h or so, meter
According to a certain concentration (generally 0.6 × 10 after number6/ml-1.0× 106/ ml) it is seeded in new triangle shaking flask, in 37 DEG C,
Continue to cultivate under conditions of 120-130r/min, clone 10 generation of forward pass.
The Colony Culture of 2.BHK-21 cells：Unicellular gram of BHK-21 is carried out in 96 orifice plates using limiting dilution assay
It is grand, 10 cells/mls are diluted to after cell count, the cell diluted is laid on 96 orifice plates, 0.1ml/ holes, 37 DEG C of 5%CO2
Culture in incubator, is observed 7.Treat that cell growth to bottom hole 80%, expands culture.Expansion order for the orifice plate of 96 orifice plates → 48 →
24 orifice plates → 6 orifice plates → 50ml triangle shaking flasks.
3.BHK-21 cell monoclonals are further subcloned：Use above limiting dilution assay, to the monoclonal cell of acquisition
3 subclones are carried out, monoclonal strain is chosen and is enlarged culture, the cell of undergrowth is eliminated during passage expands culture
Strain, by the BHK-21 monoclonal cell strains of acquisition.
The identification of 4.BHK-21 clonal cell lines：Each the 15th generation of clonal cell line is taken, triangle shaking flask is inoculated in, every 8
Hour takes each cell line to be counted, and observes the thin of each cell line, born of the same parents' size, form, when continuously observation 48 is small, chooses cell
Form is normal, and growth performance is stable, suspend the culture faster cell line of the speed of growth, is determined as BHK-21 cells Gs cells
Strain, is referred to as BHK-21-Gs cell clones, preservation information：Preservation date is on November 10th, 2017, deposit number CGMCC
No.14735。
The culture and passage of 2 BHK-21-Gs cell clones of embodiment
1st, the recovery of BHK-21-Gs cell lines：The cell line that recovery freezes, with the CD21 containing 3% (V/V) hyclone
Culture is based on cultivating in 37 DEG C of constant-temperature tables, shaking speed 120-130r/min, to cell growth to normal multiplication rate, from
The heart abandons serum, be changed to the CD21 cultures of serum-free based on continue in 37 DEG C of constant-temperature tables culture 48 it is small when, the BHK- that is recovered
21-Gs cells.
2nd, small-scale suspend culture and the passage of BHK-21-Gs cell lines：By the BHK-21 cell counts of culture that suspend
Afterwards, it is diluted to a certain concentration (generally 0.6 × 10 with cell culture fluid6/ml-1.0×106/ ml) after, it is seeded to new triangle
In shaking flask, in 37 DEG C, continue to cultivate under conditions of 120-130r/min.In incubation, to the cells of different stages of growth into
Row sampling, measures Apoptosis situation and cell viability on corresponding time point.
Apoptosis and cell viability assay method (PI decoration methods) are as follows：
(1) collect and (such as use Vial with the cell after medicine (or lytic agent) processing a period of time, measure cell concentration
1-cassette is counted)；
(2) (Optimization Steps) 400g centrifuges 5min (1000r/min), abandons supernatant, is resuspended and precipitated with 300 μ L PBS；
(3) 400g centrifuges 5min (1000r/min)；
(4) supernatant is abandoned：Carefully drawn with pipettor, not sop up precipitation, in order to avoid lose cell；
(5) cell is resuspended with Annexin V binding buffer：With 100 μ L Annexin V binding
2-4 × 10 are resuspended in buffer5Cell.It is recommended that using proper volume, make cell density in analyst coverage, so as in sample room
Carry out the comparison of result.
(6) 2 μ L Annexin V-FITC conjugates (working concentration) are added；
(7) add 4 μ L Hoechst 33342 (200 μ g/ml), mixed with pipettor；
(8) 37 DEG C of incubation 15min, pay attention to：This step is very crucial, strict temperature control and time.
(9) 400g room temperatures centrifugation 5min (1000r/min), supernatant is removed with pipettor；
Precipitation, the centrifugation of 400g (1000r/min) room temperature is resuspended in (10) 300 μ L Annexin V binding buffer
5min, supernatant is removed with pipettor；
(11) repeat step (10)；
(12) (2 μ L 500 are added with the 100 μ L Annexin V binding buffer containing 10 μ g/ml PI dyestuffs
The PI dyestuffs of μ g/ml can obtain) precipitation is resuspended, upper machine analysis detection immediately.
Measurement result shows that 48 hour cell density of culture reach 8.0 × 106/ml-1.2×107/ml；Cell viability exists
More than 91%, 72 hour cell density of culture reach 1.5 × 107/ml-2.0×107/ ml, cell viability is more than 92%；Training
Support 96 hour cell vigor and be still in more than 80%.
3rd, suspension culture of the BHK-21-Gs cell lines in 10L bioreactors：
The target cell for growing to exponential phase is subjected to normal rates amplification according to cell quantity needed for 10L reactors
Passage, continues to cultivate until adding in reactor after cell Proliferation.Condition of culture is：37 DEG C, pH7.0-7.2, dissolved oxygen 50%-
65%, rotating speed 60-120r/min, cultivate 48-60h, and cell density can increase to 8.0 × 106/ml-1.2×107/ml。
The inoculation and breeding of 3 pseudorabies virus of embodiment (Strain Ea)
1st, the preparation of kind poison：After Pseudorabies Virus Ea Strain is carried out doubling dilution with viral growth liquid, with 1 ‰ ratios
Inoculation has grown up to the BHK-21 cells of individual layer, is placed in 37 DEG C, 5% CO2In incubator, 24-72h is cultivated, treats that 75% cell occurs
Stop culture after lesion, harvested after multigelation 3 times, 12000r/min centrifugation 10min, take supernatant, measure the TCID of virus liquid50
Value, be stored in after packing -20 DEG C it is spare.
2nd, the inoculation of 10L bioreactors：Treat in cell culture fluid bhk cell density up to 7.0 × 106/ml-1.0×
107Pseudorabies virus is inoculated with after/ml, according to the ratio between virus and total number of cells 1:100 are inoculated with, and are suspended in 37 DEG C of constant temperature
48-72h is cultivated, stops culture after lesion occurs in 75% cell, is harvested after multigelation 3 times, 12000r/min centrifugations
10min, takes supernatant, measures the TCID of virus liquid50Value, be stored in after packing -80 DEG C it is spare.
The TCID of gained virus liquid50Value is 108TCID50/ml-109TCID50It is qualified to examine in the range of/ml, protected after packing
Be stored in -80 DEG C it is spare.
The inoculation and breeding of 4 foot and mouth disease virus of embodiment (OS99 plants)
1st, the preparation of kind poison：After foot and mouth disease virus (OS99 plants) is carried out doubling dilution with viral growth liquid, with
1000TCID50Ratio inoculation has been grown in the BHK-21-Gs suspending cell culture bottles of exponential phase, is placed in 37 DEG C of culture 11-
16h, stops culture after lesion occurs in 95% cell, is harvested after multigelation 3 times, and 12000r/min centrifugation 10min, take
Clearly, the TCID of virus liquid is measured50Value, be stored in after packing -80 DEG C it is spare.
2nd, the inoculation of 10L bioreactors：Treat in cell culture fluid bhk cell density up to 7.0 × 106/ml-1.0×
107Foot and mouth disease virus is inoculated with after/ml, according to the ratio between virus and total number of cells 105TCID50Ratio is inoculated with, in 37 DEG C of constant temperature
Suspend culture 11-16h, stops cultivating and harvesting after lesion occurs in 95% cell, and 12000r/min centrifugation 10min, take supernatant,
Measure the TCID of virus liquid50Value, be stored in after packing -80 DEG C it is spare.
The TCID of gained virus liquid50Value is 108TCID50/ml-109TCID50It is qualified to examine in the range of/ml, protected after packing
Be stored in -80 DEG C it is spare.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (7)
1. one plant of BHK-21 cells Gs clone strain, it is characterised in that its deposit number is 14735.
2. the suspension culture method of the BHK-21 cells Gs clone strains described in claim 1, it is characterised in that by the BHK-
21 cell line Gs clone strains are seeded to cell culture fluid, cell suspension cultures are carried out in bioreactor, condition of culture is：37
DEG C, pH7.0-7.2, dissolved oxygen amount 65%, rotating speed 60-120r/min, cultivates 48-60h；
Wherein, the cell culture fluid is the CD21 fluid nutrient mediums of pH 7.0-7.2.
3. the answering in extensive suspension culture prepares animal virus of the BHK-21 cells Gs clone strains described in claim 1
With.
4. application according to claim 3, it is characterised in that the animal virus is cell sensitive dynamic for BHK-21
Thing virus.
5. application according to claim 4, it is characterised in that the virus is foot and mouth disease virus or pseudorabies virus.
6. application according to claim 5, it is characterised in that the pseudorabies virus is by the ratio between virus and total number of cells
For 1:1000-1:100 ratio is inoculated with.
7. application according to claim 5, it is characterised in that the foot and mouth disease virus is with the ratio between total number of cells by virus
The ratio of ten a ten thousandths is inoculated with.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110157659A (en) * | 2019-05-28 | 2019-08-23 | 中牧实业股份有限公司 | A kind of BHK-21 cells BHK-21-BP-2 clone strain and its application in full suspension cell culture |
| CN114181890A (en) * | 2021-11-30 | 2022-03-15 | 中农威特生物科技股份有限公司 | Baby hamster kidney cell BHK-21-E-200 and application thereof as virus vaccine production cell strain |
| CN114317406A (en) * | 2021-12-30 | 2022-04-12 | 内蒙古必威安泰生物科技有限公司 | A kind of BHK21 suspension cell clone with strong adaptability to the growth of foot-and-mouth disease marker virus and its screening method |
| CN115521902A (en) * | 2022-10-14 | 2022-12-27 | 广州斯坦达生物科技有限公司 | Cell line for improving virus production capacity and application thereof |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0953633A1 (en) * | 1998-04-28 | 1999-11-03 | Livercell L.L.C. | Cell culturing method and medium for producing proliferated, normal, differentiated human liver cells |
| US20060148074A1 (en) * | 1996-08-30 | 2006-07-06 | Invitrogen Corporation | Serum-free mammalian cell culture medium, and uses thereof |
| CN103087995A (en) * | 2012-12-28 | 2013-05-08 | 杭州国牧生物科技有限公司 | Method of preparing foot-and-mouth disease vaccine by using BHK-21 adherent cells |
| CN106834209A (en) * | 2017-01-22 | 2017-06-13 | 西北民族大学 | The BHK21 cell clones of high density suspension culture |
| CN106916780A (en) * | 2017-01-22 | 2017-07-04 | 西北民族大学 | The BHK21 cell clones of high density suspension culture |
| CN107267466A (en) * | 2017-07-04 | 2017-10-20 | 武汉科前生物股份有限公司 | A kind of method for mass producing swine pseudorabies vaccine |
-
2017
- 2017-11-22 CN CN201711174802.4A patent/CN107988143A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20060148074A1 (en) * | 1996-08-30 | 2006-07-06 | Invitrogen Corporation | Serum-free mammalian cell culture medium, and uses thereof |
| EP0953633A1 (en) * | 1998-04-28 | 1999-11-03 | Livercell L.L.C. | Cell culturing method and medium for producing proliferated, normal, differentiated human liver cells |
| CN103087995A (en) * | 2012-12-28 | 2013-05-08 | 杭州国牧生物科技有限公司 | Method of preparing foot-and-mouth disease vaccine by using BHK-21 adherent cells |
| CN106834209A (en) * | 2017-01-22 | 2017-06-13 | 西北民族大学 | The BHK21 cell clones of high density suspension culture |
| CN106916780A (en) * | 2017-01-22 | 2017-07-04 | 西北民族大学 | The BHK21 cell clones of high density suspension culture |
| CN107267466A (en) * | 2017-07-04 | 2017-10-20 | 武汉科前生物股份有限公司 | A kind of method for mass producing swine pseudorabies vaccine |
Non-Patent Citations (5)
| Title |
|---|
| 万玉林: "低血清培养技术在口蹄疫疫苗生产中的应用研究", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
| 佛生福 等: "我国BHK-21细胞悬浮培养生产口蹄疫疫苗的进展", 《西北民族大学学报（自然科学版）》 * |
| 佛生福: "BHK-21悬浮细胞高密度克隆株的筛选及培养优化", 《中国优秀硕士学位论文全文数据库 基础科学辑》 * |
| 宁宜宝: "《兽用疫苗学》", 30 November 2008, 中国农业出版社 * |
| 王大伟 等: "猪伪狂犬病毒的增殖、纯化和鉴定", 《河南农业科学》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110157659A (en) * | 2019-05-28 | 2019-08-23 | 中牧实业股份有限公司 | A kind of BHK-21 cells BHK-21-BP-2 clone strain and its application in full suspension cell culture |
| CN110157659B (en) * | 2019-05-28 | 2021-02-09 | 中牧实业股份有限公司 | BHK-21 cell line BHK-21-BP-2 clone strain and application thereof in full suspension cell culture |
| CN114181890A (en) * | 2021-11-30 | 2022-03-15 | 中农威特生物科技股份有限公司 | Baby hamster kidney cell BHK-21-E-200 and application thereof as virus vaccine production cell strain |
| CN114181890B (en) * | 2021-11-30 | 2023-07-25 | 中农威特生物科技股份有限公司 | Milk hamster kidney cell BHK-21-E-200 and application thereof as virus vaccine production cell strain |
| CN114317406A (en) * | 2021-12-30 | 2022-04-12 | 内蒙古必威安泰生物科技有限公司 | A kind of BHK21 suspension cell clone with strong adaptability to the growth of foot-and-mouth disease marker virus and its screening method |
| CN115521902A (en) * | 2022-10-14 | 2022-12-27 | 广州斯坦达生物科技有限公司 | Cell line for improving virus production capacity and application thereof |
| CN115521902B (en) * | 2022-10-14 | 2023-04-21 | 广州斯坦达生物科技有限公司 | Cell line for improving virus production capacity and application thereof |
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