CN104232563A - Culturing method of porcine circovirus type 2-containing cells - Google Patents
Culturing method of porcine circovirus type 2-containing cells Download PDFInfo
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- CN104232563A CN104232563A CN201310241711.3A CN201310241711A CN104232563A CN 104232563 A CN104232563 A CN 104232563A CN 201310241711 A CN201310241711 A CN 201310241711A CN 104232563 A CN104232563 A CN 104232563A
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Abstract
The invention relates to a culturing method of porcine circovirus type 2-containing cells. The method comprises the following steps: cloning of an individual porcine circovirus type 2-containing PK15 cell and culturing of a subgroup of the PK15 cell: inoculating PCV2/SN07-12 seed virus into porcine circovirus type 1 pollution-free porcine kidney cells (PK15 cells), screening positive porcine circovirus type 2-containing PK15 cell cloning strains, and multiplying high-titer porcine circovirus type 2 to achieve virus titer up to 10<7.0>TCID50/ml, wherein the microbial collection registration number of the PCV2/SN07-12 seed virus is CCTCC NO:V201304. Compared with the prior art, the cells cultured by adopting the method have the advantages of being simple in technology, high in virus titer, stable in biological characteristics, and the like.
Description
Technical field
The invention belongs to the cultural method of virocyte, especially relate to the cultural method containing porcine circovirus 2 type cell.
Background technology
With porcine circovirus 2 type (Porcine Circovirus type2, PCV2) for the disease that major virulent factor causes is referred to as Porcine circovirus desease (PCV2-associated disease, PCVAD).At present, PCVAD is Occurrence & epidemic in worldwide, is one of severe infection disease of harm Swine Production.The relative disease caused for PCV2 there is no effective preventive measures, and the vaccine using PCV2 antigen to make wins initial success in the prevention of Porcine circovirus desease.Along with the Fashion and Evolution of the related vaccines caused for PCV2, diagnostic reagent and methods for the treatment of, method effectively is reliably needed to obtain enough PCV2 virus.At present, the cultivation of PCV2 is all based upon in pig kidney PK15 clone.Because PCV2 vitro culture does not produce cytopathy, virus multiplication ability, viral titer is low, and viral level is usually less than 10
5.0tCID
50/ ml, therefore the viral toxic amount of this cultural method is difficult to meet the requirement preparing vaccine.
The research of domestic Duo Jia unit, by carrying out limiting dilution and cell clone to PK15 parent cell, obtains clone PCV2 to hypersensitivity, and the report that the clone of other type carries out limiting dilution and cell clone is quite a few.Research main technological route at present about this viral titer is the multiplication capacity adopting D-glucosamine process cell enhanced virus, or adopts virus to go down to posterity in cell cultured continuously, and make strain isolated increasing progressively with passage number of times, virus titer raises to some extent; But the lead time is long, complex process, the more difficult control of quality product, cost is higher, is unfavorable for the vaccine of large-scale production reasonable price.At present, there is no the report carrying out cultivating porcine circovirus 2 type by carrying out limiting dilution and cell clone containing the PK15 cell of porcine circovirus 2 type both at home and abroad.
Summary of the invention
Object of the present invention be exactly provide that a kind of technique is simple, virus titer is high to overcome defect that above-mentioned prior art exists, the cultural method containing porcine circovirus 2 type cell that biological characteristics is stable.
Object of the present invention can be achieved through the following technical solutions:
Containing the cultural method of porcine circovirus 2 type cell, comprise the following steps:
(1) the single cultivation containing porcine circovirus 2 type PK15 cell clone and subgroup thereof
By PCV2/SN07-12 kind poison be inoculated in without 1 type pig circular ring virus pollute porcine kidney cell (PK15 cell) in and cultivate in 37 DEG C of incubators, trysinization is used to be in the PK15 cell of individual layer containing porcine circovirus 2 type of logarithmic phase, then limiting dilution assay is adopted, according to every hole 100uL containing 20wt% (please amendment be audit) bovine serum cell growth medium, the density of 0.5 cells/well, adds 96 porocyte culture plates and is placed in containing 5vt%CO
2, temperature is cultivate in 37 DEG C of incubators, selects cell in the cell hole of individual cells growth and carries out subclone cultivation, use after trysinization, add bovine serum cell growth medium, then adds 96 porocyte culture plates and be placed in containing 5vt%CO
2, temperature is cultivate 72h in 37 DEG C of incubators;
(2) the positive screening containing porcine circovirus 2 type PK15 cell clone
Get and cover with individual layer 96 porocyte culture plate, discard nutrient solution, PBS washed cell 3 times, through dehydrated alcohol at-20 DEG C of fixing 2h, confining liquid washs 1 time, recycling confining liquid is at 37 DEG C of effect 30min, add PCV2 antiserum(antisera), 37 DEG C of effect 45min, after PBS washed cell 3 times, add fluorescent mark A albumen (FITC-SPA), 37 DEG C of effect 45min, with mounting liquid (Mounting-Fluid) after washing 3 times, in fluorescence microscopy Microscopic observation specificity fluorescent cell, not connect the normal PK15 cell of poison as negative control, selecting the maximum clone strain of amount of fluorescence is the high infective PK15 cell subsets of porcine circovirus 2 type, repeated cloning once, every time cloning all needs to do infection proportion and detects, the PK15-Ag clone containing PCV2 height infection proportion is obtained to carry out purifying containing porcine circovirus 2 type PK15 cell strain after.
(3) propagation of high titre porcine circovirus 2 type
Trysinization is utilized to contain the PK15-A8 clone of PCV2 height infection proportion and be inoculated in Tissue Culture Flask, add the DMEM nutritive medium containing 7wt%-10wt% calf serum, 24h is cultivated at 37 DEG C, abandon growth media, add the DMEM maintenance medium containing 2wt% calf serum, cultivate 72-96h in 37 DEG C, harvested cell culture, freeze thawing 3 times, had both obtained high titre porcine circovirus 2 type, and the virus titer that IFA method measures reaches 10
7.0tCID
50/ ml.
PCV2/SN07-12 kind poison described in step (1) is the microbial preservation registration number of Circovirus porcine circovirus 2 type (Porcine Circovirus Type2): CCTCC NO:V201304 its microbial preservation number is: CCTCC NO:V201114; The preservation time: 2013.3.15; Depositary institution: China typical culture collection center, CHINA CENTER FOR TYPE CULTURE COLLECTION (CCTCC); Preservation address: Wuhan University's preservation center, Wuchang District, Wuhan City, Hubei Province (affiliated primary school of Wuhan University first opposite).
In step (2), the PK15-A8 cell obtained containing PCV2 height infection proportion lies in-196 DEG C of preservations.
Compared with prior art, the present invention has the following advantages:
(1) technique is simple: plant cell expansion and cultivate harvest liquid and be the stoste of vaccine, save the malicious rejuvenation of kind in production technique compared with conventional P CV2 vaccine preparation method, virus inoculation adsorbs and adopt the techniques such as D-glucosamine process cell;
(2) virus titer is high: virus titer reaches 10
7.0tCID
50/ ml.
(3) biological characteristics is stablized: porcine circovirus 2 type is stablized at PK15-A8 cellular proliferative, pollutes without inoculating microbe.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in detail.
Embodiment
Containing the cultural method of porcine circovirus 2 type cell, comprise the following steps:
(1) the single cultivation containing porcine circovirus 2 type PK15 cell clone and subgroup thereof
By PCV2/SN07-12 kind poison be inoculated in without 1 type pig circular ring virus pollute porcine kidney cell (PK15 cell) in and cultivate in 37 DEG C of incubators, trysinization is used to be in the PK15 cell of individual layer containing porcine circovirus 2 type of logarithmic phase, then limiting dilution assay is adopted, according to every hole 100uL containing 20wt% bovine serum cell growth medium, the density of 0.5 cells/well, adds 96 porocyte culture plates and is placed in containing 5vt%CO
2, temperature is cultivate in 37 DEG C of incubators, selects cell in the cell hole of individual cells growth and carries out subclone cultivation, use after trysinization, add bovine serum cell growth medium, then adds 96 porocyte culture plates and be placed in containing 5moL/LCO
2, temperature is cultivate 72h in 37 DEG C of incubators.
(2) the positive screening containing porcine circovirus 2 type PK15 cell clone
Get and cover with individual layer 96 porocyte culture plate, discard nutrient solution, PBS washed cell 3 times, through dehydrated alcohol at-20 DEG C of fixing 2h, confining liquid washs 1 time, recycling confining liquid is at 37 DEG C of effect 30min, add PCV2 antiserum(antisera), 37 DEG C of effect 45min, after PBS washed cell 3 times, add fluorescent mark A albumen (FITC-SPA), 37 DEG C of effect 45min, with mounting liquid (Mounting-Fluid) after washing 3 times, in fluorescence microscopy Microscopic observation specificity fluorescent cell, not connect the normal PK15 cell of poison as negative control, selecting the maximum clone strain of amount of fluorescence is the high infective PK15 cell subsets of porcine circovirus 2 type, repeated cloning once, every time cloning all needs to do infection proportion and detects, the PK15-A8 clone containing PCV2 height infection proportion is obtained to carry out purifying containing porcine circovirus 2 type PK15 cell strain after,-196 DEG C of preservations.
(3) enrichment procedure of high titre porcine circovirus 2 type
Trysinization is utilized to contain the PK15-A8 clone of PCV2 height infection proportion and be inoculated in Tissue Culture Flask, add the DMEM nutritive medium containing 7wt%-10wt% calf serum, 24h is cultivated at 37 DEG C, abandon growth media, add the DMEM maintenance medium containing 2wt% calf serum, cultivate 72-96h in 37 DEG C, harvested cell culture, freeze thawing 3 times, had both obtained high titre porcine circovirus 2 type, and the virus titer that IFA method measures reaches 10
7.0tCID
50/ ml.
To the characteristic research containing high titre porcine circovirus 2 type PK15-A8 clone
1, the research of cellular form
Get equivalent without porcine circovirus 2 type PK15 cell, be inoculated in 24 orifice plates respectively containing porcine circovirus 2 type PK15-A8 clone strain cell, add the DMEM nutrient solution of 7wt%-10wt% calf serum, cultivate 48 hours, treat that cell covers with individual layer and observes its morphological differences for 37 DEG C.Two strain cellular form differences are not remarkable.
2, the research of vitro growth rates
Get 1 × 10
5individual cell is inoculated in 6 orifice plates and cultivates, and adds the DMEM nutrient solution containing 7wt%-10wt% calf serum, 37 DEG C of cultivations, gets wherein 1 porocyte count respectively at 12h, 24h, 36h and 48h.With compared with porcine circovirus 2 type PK15 cell, PK15-A8 vitro growth rates is relatively slack-off.
3, the research of cytobiology stability
By the PK15-A8 passage containing porcine circovirus 2 type after colony screening to the 40th generation, choose the 5th, 10,20,30,40 generation cell be laid on 96 orifice plates, after 37 DEG C of cultivation 72-96h, amount of fluorescence is detected by IFA method, observe the difference of amount of fluorescence between different generation, to judge whether the virus of the PK15-A8 cell proliferation containing the porcine circovirus 2 type characteristic of cloning is stablized.IFA measurement result shows that porcine circovirus 2 type is stablized at PK15-A8 cellular proliferative.
4, the research of cell external source Viral diagnosis
Get the 5th, 10,20,30,40 generations containing the PK15-A8 cell of porcine circovirus 2 type, extract the virus total RNA in culture, the viruses such as PCV1, HCV, PPV, BVDV and PRV detected, set known viruse contrast simultaneously.Result shows, and other exogenous virus is feminine gender.
5, the research of cell detection of mycoplasma
Get the 5th, 10,20,30,40 generations containing the PK15-A8 cell of porcine circovirus 2 type, by the method for " Chinese veterinary pharmacopoeia " annex, get cell conditioned medium, be inoculated in mycoplasma culture medium in proportion, set up negative control simultaneously, cultivate 5-7 days in 37 DEG C.Detected result shows that this clone strain is without mycoplasma contamination.
6, the research of PCV2 growth curve in clonal cell line
Get 1 × 10
5individual cell is inoculated in 6 porocyte plates, and 24h, 48h, 72h, 96h and 120h harvested cell supernatant measures viral TCID by IFA method
50, repeat 3 times.Result shows, containing the PK15-A8 cell cultures of porcine circovirus 2 type to 72-96 hour, viral level is the highest.
Claims (3)
1., containing the cultural method of porcine circovirus 2 type cell, it is characterized in that, comprise the following steps:
(1) the single cultivation containing porcine circovirus 2 type PK15 cell clone and subgroup thereof
By PCV2/SN07-12 kind poison be inoculated in without 1 type pig circular ring virus pollute porcine kidney cell (PK15 cell) in and cultivate in 37 DEG C of incubators, trysinization is used to be in the PK15 cell of individual layer containing porcine circovirus 2 type of logarithmic phase, then limiting dilution assay is adopted, according to every hole 100uL containing 20wt% bovine serum cell growth medium, the density of 0.5 cells/well, adds 96 porocyte culture plates and is placed in containing 5vt%CO
2, temperature is cultivate in 37 DEG C of incubators, selects cell in the cell hole of individual cells growth and carries out subclone cultivation, use after trysinization, add bovine serum cell growth medium, then adds 96 porocyte culture plates and be placed in containing 5vt%CO
2, temperature is cultivate 72h in 37 DEG C of incubators;
(2) the positive screening containing porcine circovirus 2 type PK15 cell clone
Get and cover with individual layer 96 porocyte culture plate, discard nutrient solution, PBS washed cell 3 times, through dehydrated alcohol at-20 DEG C of fixing 2h, confining liquid washs 1 time, recycling confining liquid is at 37 DEG C of effect 30min, add PCV2 antiserum(antisera), 37 DEG C of effect 45min, after PBS washed cell 3 times, add fluorescent mark A albumen (FITC-SPA), 37 DEG C of effect 45min, with mounting liquid (Mounting-Fluid) after washing 3 times, selecting the maximum clone strain of amount of fluorescence under fluorescent microscope is the high infective PK15 cell subsets of porcine circovirus 2 type, repeated cloning once, the PK15-A8 clone containing PCV2 height infection proportion is obtained to carry out purifying containing porcine circovirus 2 type PK15 cell strain after,
(3) propagation of high titre porcine circovirus 2 type
Trysinization is utilized to contain the PK15-A8 clone of PCV2 height infection proportion and be inoculated in Tissue Culture Flask, add the DMEM nutritive medium containing 7wt%-10wt% calf serum, 24h is cultivated at 37 DEG C, abandon growth media, add the DMEM maintenance medium containing 2wt% calf serum, cultivate 72-96h in 37 DEG C, harvested cell culture, freeze thawing 3 times, had both obtained high titre porcine circovirus 2 type.
2. the cultural method containing porcine circovirus 2 type cell according to claim 1, it is characterized in that, the malicious microbial preservation registration number for Circovirus porcine circovirus 2 type (Porcine Circovirus Type2) of the PCV2/SN07-12 kind described in step (1) is: CCTCC NO:V201304.
3. the cultural method containing porcine circovirus 2 type cell according to claim 1, is characterized in that, in step (2), the PK15-A8 cell obtained containing PCV2 height infection proportion lies in-196 DEG C of preservations.
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| CN107475205A (en) * | 2017-08-31 | 2017-12-15 | 浙江美保龙生物技术有限公司 | A kind of pig circular ring virus separation method |
| CN113406337A (en) * | 2021-06-23 | 2021-09-17 | 金宇保灵生物药品有限公司 | Screening method of Marc-145 cell strain capable of being used for culturing PRRSV to obtain virus liquid with high virus content |
| CN113804522A (en) * | 2021-08-20 | 2021-12-17 | 北京英诺特生物技术股份有限公司 | Method for preparing cell substrate, method for preparing kit and joint inspection method |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106754747A (en) * | 2016-12-06 | 2017-05-31 | 武汉市工程科学技术研究院 | New pig circular ring virus and its co-cultured cell strain |
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| CN107475205A (en) * | 2017-08-31 | 2017-12-15 | 浙江美保龙生物技术有限公司 | A kind of pig circular ring virus separation method |
| CN113406337A (en) * | 2021-06-23 | 2021-09-17 | 金宇保灵生物药品有限公司 | Screening method of Marc-145 cell strain capable of being used for culturing PRRSV to obtain virus liquid with high virus content |
| CN113804522A (en) * | 2021-08-20 | 2021-12-17 | 北京英诺特生物技术股份有限公司 | Method for preparing cell substrate, method for preparing kit and joint inspection method |
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Application publication date: 20141224 |