CN109810952A - The method of human respiratory syncytial virus's large-scale culture - Google Patents
The method of human respiratory syncytial virus's large-scale culture Download PDFInfo
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- CN109810952A CN109810952A CN201910288101.6A CN201910288101A CN109810952A CN 109810952 A CN109810952 A CN 109810952A CN 201910288101 A CN201910288101 A CN 201910288101A CN 109810952 A CN109810952 A CN 109810952A
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Abstract
The invention discloses a kind of methods of human respiratory syncytial virus's large-scale culture, first digest the Hep-2 cell for covering with single layer using pancreatin digestive juice, then add cell suspension and expand on a large scale in cell factory progress cell；After bottom covers with single layer Hep-2 cell, human respiratory syncytial virus is inoculated in cell factory, culture medium is changed to serum free medium and is cultivated within first day after inoculation, daily using virus titer in ELISA method detection supernatant, draws virus multiplication curve；Culture reach peak to virus titer after, collect the Virus culture supernatant of each bottle, with batch merge after, set 2-8 DEG C it is spare.In the method for the present invention, culture medium is changed to serum free medium within first day after virus inoculation, the step of greatly reducing the content for harvesting calf serum in virus liquid, reducing downstream purification, provides guarantee for the development of people's respiratory syncytial virus vaccines.
Description
Technical field
The present invention relates to the cultures of virus, more particularly, to a kind of method of human respiratory syncytial virus's large-scale culture.
Background technique
Human respiratory syncytial virus (RSV) belongs to Paramyxoviridae, which propagates through airborne droplet and close contact,
It is more common in newborn and the baby within 6 months, incubation period 3 ~ 7 days.Infant's symptom is heavier, is presented with high fever, rhinitis, pharyngitis
And laryngitis, capillary bronchitis and pneumonia are shown as later；A small number of sick children can concurrent tympanitis, pleurisy and myocarditis etc..It is adult
After older children infection, it is mainly shown as the infection of the upper respiratory tract.Make a definite diagnosis separable virus and do serum complement combine test and
Neutralization test.Viral antigen in nasopharyngeal secretions is checked using immunofluorescence technique, can make quick diagnosis.Treatment is to support and right
Based on disease therapy, when having secondary bacterial infection, it can be treated with antimicrobial.
RSV finding under Electronic Speculum is similar with parainfluenza virus, and virion size is about 150nm, slightly compared with parainfluenza virus
It is small, it is RNA virus, no hemagglutination sensitive to ether forms distinctive conjunction born of the same parents in people's epithelial tissue culture, and virus is in born of the same parents
Proliferation in slurry, it is seen that intracytoplasmic inclusion.
Up to now, for respiratory tract infection caused by human airway syncytial virus, there are no specific medicaments, also do not have
Produce vaccine.And vaccine is manufactured, large-scale culture human airway syncytial virus is just needed first.Tradition culture human breathing
The method of road syncytial virus is: cell is passed on amplification by glass roller bottles step by step, after virus inoculation, through culture in 3 ~ 7 days, then
Viral suspension harvest is carried out again.Due to containing 2% calf serum in virus inoculation culture medium, later-period purification there are larger difficulty,
If being used for preparing vaccine, increase the risk that cow's serum residual is more than national standard；It is produced simultaneously using glass roller bottles
Human airway syncytial virus, the bottle quantity needed is more, complicated for operation, high labor cost, has seriously affected to human breathing
The further research of road syncytial virus vaccines.
Summary of the invention
The purpose of the present invention is to provide a kind of easy to operate, large-scale culture can be carried out to human respiratory syncytial virus
Method, to meet the research and manufacture demand to human airway syncytial virus vaccines.
To achieve the above object, the present invention can take following technical proposals:
The method of human respiratory syncytial virus's large-scale culture of the present invention, including following specific steps:
The first step, will cover with the Hep-2 cell of single layer using pancreatin digestive juice vitellophag in culture bottle, then by 1:3-4 into
The passage of row cell expands, in 37 DEG C, 5%CO2Under the conditions of cultivate；
Second step, using cell factory, the cell suspension obtained after the first step is digested carries out cell by 200ml/ layers of additive amount
Extensive amplification；
Third step, by human respiratory syncytial virus by 0.01 ~ 0.1MOI be inoculated in cell factory bottom cover with single layer Hep-2 it is thin
On born of the same parents, using the DMEM culture medium containing 2% calf serum, in 34 ± 1 DEG C, 5%CO2Under the conditions of cultivate；
4th step carries out changing liquid, culture medium is changed to serum free medium, then in 34 ± 1 DEG C, 5% for first day after inoculation
CO2Under the conditions of cultivate；In incubation, daily using virus titer in ELISA method detection supernatant, virus multiplication curve is drawn；
5th step collects each bottle after culture to virus titer reaches peak (ELISA method detection light absorption value is not less than 3.5)
Virus culture supernatant, with batch merge after, set 2-8 DEG C it is spare.
4th step changes the serum substitute that serum free medium used after liquid is the addition 1% in DMEM culture medium
(ITSE of such as Sigma) is prepared.
The advantage of the invention is that first day after virus inoculation carries out changing liquid, culture medium is changed to the nothing after improvement
The step of blood serum medium greatly reduces the content of calf serum in harvest virus liquid, reduces downstream purification, behaviour is exhaled
Cow's serum residual in road syncytial virus vaccines is inhaled to meet national standard and provide guarantee.
Specific embodiment
Below by specific example, more detailed explanation is made to the method for the present invention, in order to those skilled in the art couple
The understanding of the application.Reagent used in the present invention and instrument are this field conventional products, and detection method used is also this field
Conventional method.
The method of human respiratory syncytial virus's large-scale culture of the present invention, including following specific steps:
The first step selects 125cm2Hep-2 cell is inoculated in culture bottle (generally 50ml/ in the ratio of 1:3-4 by square vase
Bottle), after Hep-2 cell covers with single layer in culture bottle, using pancreatin digestive juice vitellophag, after cell is at ground-glass appearance,
The culture medium containing calf serum is added, terminates digestion, has hanged cell；Then it is expanded in the passage that the ratio of 1:3-4 carries out cell,
Culture bottle is put into 37 DEG C, 5%CO in incubator2Under the conditions of cultivate 3 days；
Second step selects 10 layer cell factories (in actual use, can be adjusted according to required scale according to the method for the first step
It is whole) it carries out cell and expands on a large scale, every layer is added 200ml and contains the DMEM culture medium of 10% calf serum；
Third step after the cell after expand on a large scale covers with single layer, by human respiratory syncytial virus in 37 DEG C of fast melts, is pressed
0.05MOI calculates virus inoculation amount, and the seed culture of viruses of required volume is inoculated on Hep-2 cell, and is added containing 2% calf serum
DMEM culture medium 200ml to every layer cell factory, in 34 ± 1 DEG C, 5%CO2Culture；
4th step carries out changing liquid for first day after inoculation, after DMEM culture medium of the original containing 2% calf serum is changed to improvement
Serum free medium (DMEM containing 1% serum substitute ITSE)；
5th step, next aseptic aspiration culture supernatant is drawn using virus titer in ELISA sandwich method detection supernatant daily
Virus multiplication curve, see the table below 1；
Progress virus titer detection in the 6th day after poison is connect, enzyme exempts from light absorption value and reaches 6.39, collects the Virus culture supernatant of each bottle, together
Virus-culturing fluid after batch merging is placed in 2-8 DEG C and saves backup.
Through detecting, the cow's serum residual quantity in virus-culturing fluid is 39.6ng/ml, meets national standard (not higher than 50ng/ml)
Regulation.
The method of the present invention is easy to operate, can carry out large-scale culture to human respiratory syncytial virus, prepare high-titer
Viral cultures meet the further research to human airway syncytial virus vaccines.
Claims (2)
1. a kind of method of human respiratory syncytial virus's large-scale culture, it is characterised in that: including following specific steps:
The first step, will cover with the Hep-2 cell of single layer using pancreatin digestive juice vitellophag in culture bottle, then by 1:3-4 into
The passage of row cell expands, in 37 DEG C, 5%CO2Under the conditions of cultivate；
Second step, using cell factory, the cell suspension obtained after the first step is digested carries out cell by 200ml/ layers of additive amount
Extensive amplification；
Third step, by human respiratory syncytial virus by 0.01 ~ 0.1MOI be inoculated in cell factory bottom cover with single layer Hep-2 it is thin
On born of the same parents, using the DMEM culture medium containing 2% calf serum, in 34 ± 1 DEG C, 5%CO2Under the conditions of cultivate；
4th step carries out changing liquid, culture medium is changed to serum free medium, then in 34 ± 1 DEG C, 5% for first day after inoculation
CO2Under the conditions of cultivate；In incubation, daily using virus titer in ELISA method detection supernatant, virus multiplication curve is drawn；
5th step collects the Virus culture supernatant of each bottle after culture reaches peak to virus titer, after merging with batch, sets
2-8 DEG C spare.
2. the method for human respiratory syncytial virus's large-scale culture according to claim 1, it is characterised in that: the described 4th
The serum substitute that serum free medium used after changing liquid is the addition 1% in DMEM culture medium is walked to be prepared.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112574961A (en) * | 2020-12-28 | 2021-03-30 | 兆丰华生物科技(南京)有限公司 | Preparation method of cell vaccine antigen |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4145252A (en) * | 1977-08-17 | 1979-03-20 | Merck & Co., Inc. | Respiratory syncytial vaccine |
| CN102559608A (en) * | 2012-01-16 | 2012-07-11 | 遵义医学院 | Method for separating and culturing RSV (Respiratory Syncytial Virus) by using OMC (Ostiomeatal Complex)-685-1 cells |
| CN109402068A (en) * | 2018-11-09 | 2019-03-01 | 四川华神兽用生物制品有限公司 | A method of preparing the remaining porcine pseudorabies virus of serum-free |
-
2019
- 2019-04-11 CN CN201910288101.6A patent/CN109810952A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4145252A (en) * | 1977-08-17 | 1979-03-20 | Merck & Co., Inc. | Respiratory syncytial vaccine |
| CN102559608A (en) * | 2012-01-16 | 2012-07-11 | 遵义医学院 | Method for separating and culturing RSV (Respiratory Syncytial Virus) by using OMC (Ostiomeatal Complex)-685-1 cells |
| CN109402068A (en) * | 2018-11-09 | 2019-03-01 | 四川华神兽用生物制品有限公司 | A method of preparing the remaining porcine pseudorabies virus of serum-free |
Non-Patent Citations (5)
| Title |
|---|
| GREGORY A. PRINCE等: "Vaccine-enhanced respiratory syncytial virus disease in cotton rats following immunization with Lot 100 or a newly prepared reference vaccine", 《JOURNAL OF GENERAL VIROLOGY》, vol. 82, 31 December 2001 (2001-12-31), pages 2882 * |
| 刘菊等: "呼吸道合胞病毒Long株的培养及应用", 《微生物学杂志》, vol. 24, no. 05, 30 September 2004 (2004-09-30), pages 47 - 49 * |
| 廖辉等: "呼吸道合胞病毒对Hep-2细胞增殖的影响", 《现代中西医结合杂志》, vol. 17, no. 12, 20 April 2008 (2008-04-20), pages 1794 - 1795 * |
| 王欣怡等: "呼吸道合胞病毒培养及其病毒滴度检测方法的建立", 《中国生物制品学杂志》, vol. 28, no. 09, 30 September 2015 (2015-09-30), pages 3 - 1 * |
| 谢忠平等: "人呼吸道合胞病毒A_2株在不同细胞中的培养及其影响因素", 《中国生物制品学杂志》, vol. 22, no. 06, 20 June 2009 (2009-06-20), pages 574 - 577 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112574961A (en) * | 2020-12-28 | 2021-03-30 | 兆丰华生物科技(南京)有限公司 | Preparation method of cell vaccine antigen |
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Application publication date: 20190528 |