CN105420198A - Application of chicken liver cancer cell system as duck plague virus host - Google Patents
Application of chicken liver cancer cell system as duck plague virus host Download PDFInfo
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- CN105420198A CN105420198A CN201510977117.XA CN201510977117A CN105420198A CN 105420198 A CN105420198 A CN 105420198A CN 201510977117 A CN201510977117 A CN 201510977117A CN 105420198 A CN105420198 A CN 105420198A
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Abstract
The invention belongs to the technical field of duck plague virus culturing, particularly relates to a new host cell of the duck plague virus, and discloses application of a chicken liver cancer cell system as a duck plague virus host. A traditional method for culturing the duck plague virus through the duck embryo fibroblast primary cell is limited by duck embryo supply and the hatching day age, and wastes time and labor during preparation. The established method for culturing the duck plague virus chicken liver cancer cell system is good in virus growth. The method for culturing the virus through a continuous cell line is not influenced by duck embryo supply, and has the advantages of being instantly available and capable of saving time and labor. The duck plague virus can grow and reproduce on a chicken liver cancer cell system continuous cell culture medium, and by means of a newly-found virus copying host cell, a new technical approach, method and material are provided for research of related fields.
Description
Technical field
The invention belongs to duck plague virus culture technique field, be specifically related to the new host cell of duck plague virus.
Background technology
Duck plague (DuckPlague, DP) be the transmissible disease of a kind of acute septic being common in the geese flying in a line-brothers order aquatic birds such as duck, goose, swan, wild goose caused by duck plague virus (DuckPlagueVirus, DPV) and high degree in contact, height lethality.This disease popularity extensively, propagation is rapid, M & M is all higher, and being to endanger one of epidemic disease the most serious to world wide aquatic bird aquaculture at present, is the formidable enemy of foster duck industry.This epidemic disease is in China and extensively distribute all over the world, and causes serious harm, so have great importance to the research of this disease.
DPV belongs to duck simplexvirus 1 type in simplexvirus first subfamily.DPV can in 9-14 age in days duck embryo chorioallantoic cavity growth and breeding, but the growth of DPV is by the impact in duck embryo source, if plant duck immunity to cross duck plague virus vaccine, plant the DPV specificity maternal antibody containing high titre in egg, then due to the neutralizing effect of virus, DPV can not grow and copy in duck embryo.DPV also can become growing multiplication in fiber-like continuous cell line CCL-141, and has the advantages that plaque is easy to observation, but viral yield comparatively few 5.6 times more than of primary cell, illustrate that virus growth and breeding is wherein too late at primary cell middle ideal.Above-mentioned cultural method is the main path obtaining DPV viral cultures at present.
The main prior art of DPV growth and breeding in DEF (DEF) is: use the duck embryo about 12 ages in days now with now preparing DEF cell.The weak point of the method shows as: 1. by the restriction of duck embryo supply, and stopping, term is available without zygote; 2. by duck embryo source impact, there are some feedings and managements and the poor plant of sanitary condition, some bacillary vertical transmission diseases, as the existence of the cause of disease such as Salmonellas, mycoplasma, easily cause the pollution of this batch of DEF primary cell, directly affect the carrying out of subsequent experimental.3. traditional method time cycle of preparing needed for cell is longer, and preparing DEF each time all needs to repeat same process, and due to kind of egg batch difference, the biological property preparing DEF each time may have difference.
Chicken gizzard cancerous cell line (LMH clone) set up in 1981 by a Japanese scholars.By using diethylnitrosamine to induce for a long time male Leghorn, cause chicken body to produce liver cancer, then isolated cell obtain from liver cancer tissue.
Summary of the invention
Technical problem to be solved by this invention is to provide the new Hosts of a kind of duck plague virus, and the correlative study for this area opens new approach and virus culture Hosts, has enriched the content of correlative study.
For solving the problems of the technologies described above, the technical solution used in the present invention is as follows:
Chicken gizzard cancerous cell line (LMH) is as the application of duck plague virus (DPV) host.
Described chicken gizzard cancerous cell line is preferably through following pre-treatment: conventionally cultivate chicken gizzard cancer cells, when cell just grows up to cell monolayer time, solid-liquid separation gets chicken gizzard cancer cells, cleans cell surface 3 times with sterilizing PBS.
Wherein, described solid-liquid separation, its means are tipping nutrient solution.
The method of chicken gizzard cancerous cell line inoculation duck plague virus is preferably: in cell bottle, duck plague virus kind venom is covered chicken gizzard cancer cell surfaces, absorption 20-30min (preferred 30min) is carried out under 37 DEG C of conditions, solid-liquid separation, abandon virus liquid, then add containing volumn concentration be the calf serum of 3% and the dual anti-of 100IU/ml DMEM maintain nutritive medium in 37 DEG C of cell culture incubators, cultivate 48-72 hour.
Wherein, comparatively preferred process, in adsorption process, shakes cell bottle gently every 5-10 (preferably 5 minutes), makes virus be uniformly distributed on cell thus fully infect.
Wherein, described solid-liquid separation is tipping supernatant liquor.
Wherein, comparatively preferred process is that duck plague virus kind venom consumption is advisable can cover cell monolayer.
Wherein, comparatively preferred process is, generally for 75cm
2in Tissue Culture Flask, the consumption that DMEM maintains nutritive medium is 25 milliliters.
Described 100IU/ml dual anti-is the combination of 100IU/ml penicillin and 100IU/ml Streptomycin sulphate.
Adopt the inventive method with chicken gizzard cancerous cell line for host inoculates duck plague virus, gather the cell (shown in Fig. 1) after inoculation duck plague virus and compared with control cells (shown in Fig. 2) respectively, make cell ultrathin section(ing), can observe and connect in poison cell, there is a large amount of duck plague virus particles, and compared with control cells is not observed, determine that DPV grows on LMH cell, chicken gizzard cancerous cell line can copy host as duck plague virus.
Beneficial effect: the inventive method compared with prior art, has following advantage:
1, traditional DEF primary cell culture duck plague virus method, be often subject to the restriction of the supply of duck embryo, hatching age in days, preparation process wastes time and energy.The duck plague virus LMH cell culture processes that the present invention sets up, viral growth is good.This continuous cell line cultivates the method for virus, does not affect, have ready access upon use, time saving and energy saving advantage by the supply of duck embryo.
2, the present invention show duck plague virus can on chicken gizzard cancerous cell line passage cell culture growth and breeding, as newfound virus replication host cell, the research for association area provides new technological approaches and method material.
Accompanying drawing explanation
Fig. 1 is the Electronic Speculum ultrathin section(ing) that in embodiment 1, duck plague virus grows on chicken gizzard cancerous cell line, and wherein M represents plastosome; N represents nucleus.
Fig. 2 is compared with control cells Electronic Speculum ultrathin section(ing) in embodiment 1.
Embodiment
According to following embodiment, the present invention may be better understood.But those skilled in the art will readily understand, the content described by embodiment only for illustration of the present invention, and should can not limit the present invention described in detail in claims yet.
In following examples, chicken gizzard cancerous cell line source (come from ATCC,
number:CRL-2117
tM).Duck plague virus is duck plague virus chicken embryo low virulent strain, for the continuous passage of this use for laboratory duck plague virus strain isolated chicken embryo obtains.
Embodiment 1:
LMH cell is cultivated according to laboratory ordinary method, when cell density reaches about 95% time, get the LMH that this has just grown up to individual layer, abandon growth nutrient solution, clean cell surface 3 times with sterilizing PBS, then the inoculation of DPV kind poison is joined LMH cell surface and inoculate.Duck plague virus liquid covers cell surface and adsorbs, and hatches centre and shakes cell bottle gently every 5 minutes, makes virus be uniformly distributed on cell thus fully infect.Abandon virus liquid after 37 DEG C of absorption 30min, the DMEM then added containing 3% calf serum and 100IU/ml penicillin and 100IU/ml Streptomycin sulphate maintains nutritive medium in 37 DEG C of cultivations, observes the change of CPE every day under inverted microscope.Fig. 1 is the Electronic Speculum ultrathin section(ing) schematic diagram that duck plague virus grows on chicken gizzard cancerous cell line, and arrow is duck plague virus, proves that duck plague virus can grow on LMH cell.
Claims (9)
1. chicken gizzard cancerous cell line is as the application of duck plague virus host.
2. application according to claim 1, is characterized in that, described chicken gizzard cancerous cell line is through following pre-treatment: cultivate chicken gizzard cancer cells, when cell grows up to cell monolayer, solid-liquid separation gets chicken gizzard cancer cells, cleans cell surface 3 times with sterilizing PBS.
3. application according to claim 1, is characterized in that, described solid-liquid separation, and its means are tipping nutrient solution.
4. application according to claim 1, it is characterized in that, the method of chicken gizzard cancerous cell line inoculation duck plague virus is: in cell bottle, duck plague virus kind venom is covered chicken gizzard cancer cell surfaces, absorption 20-30min is carried out under 37 DEG C of conditions, solid-liquid separation, abandons virus liquid, then add containing volumn concentration be 3% calf serum and the dual anti-DMEM of 100IU/ml maintain nutritive medium cultivate 48-72 hour in 37 DEG C of cell culture incubator.
5. application according to claim 4, is characterized in that, in adsorption process, shakes cell bottle gently every 5-10 minute, makes virus be uniformly distributed on cell thus fully infect.
6. application according to claim 4, is characterized in that, described solid-liquid separation is tipping supernatant liquor.
7. application according to claim 4, is characterized in that, duck plague virus kind venom consumption is for covering cell monolayer.
8. application according to claim 4, is characterized in that, 75cm
2in Tissue Culture Flask, the consumption that DMEM maintains nutritive medium is 25 milliliters.
9. application according to claim 4, is characterized in that, described 100IU/ml is dual anti-is the combination of 100IU/ml penicillin and 100IU/ml Streptomycin sulphate.
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| CN201510977117.XA CN105420198B (en) | 2015-12-23 | 2015-12-23 | Application of chicken liver cancer cell line as duck plague virus host |
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| CN201510977117.XA CN105420198B (en) | 2015-12-23 | 2015-12-23 | Application of chicken liver cancer cell line as duck plague virus host |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109207439A (en) * | 2018-10-09 | 2019-01-15 | 华农（肇庆）生物产业技术研究院有限公司 | A kind of full suspension culture method of duck plague virus |
| CN109234239A (en) * | 2018-07-13 | 2019-01-18 | 广东永顺生物制药股份有限公司 | A kind of cultural method of duck plague virus |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1202378A (en) * | 1997-06-02 | 1998-12-23 | 财团法人化学及血清疗法研究所 | Process for preparing infectious bursal disease virus and avian reovirus using cell lines |
| CN102066407A (en) * | 2008-04-28 | 2011-05-18 | 英特威国际有限公司 | Novel avian astrovirus |
-
2015
- 2015-12-23 CN CN201510977117.XA patent/CN105420198B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1202378A (en) * | 1997-06-02 | 1998-12-23 | 财团法人化学及血清疗法研究所 | Process for preparing infectious bursal disease virus and avian reovirus using cell lines |
| CN102066407A (en) * | 2008-04-28 | 2011-05-18 | 英特威国际有限公司 | Novel avian astrovirus |
Non-Patent Citations (3)
| Title |
|---|
| A. H. DARDIRI: "Attenuation of Duck Plague Virus I and Its Propagation in Cell Culture", 《ARCHLY FIIR DIE GESAMTE VIRUSFORSCHUNG》 * |
| LYNN D. CONDREAY ET AL: "Efficient Duck Hepatitis B Virus Production by an Avian Liver Tumor Cell Line", 《JOURNAL OF VIROLOGY》 * |
| TOKUICHI KAWAGUCHI ET AL: "Establishment and Characterization of a Chicken Hepatocellular Carcinoma Cell Line, LMH", 《CANCER RESEARCH》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109234239A (en) * | 2018-07-13 | 2019-01-18 | 广东永顺生物制药股份有限公司 | A kind of cultural method of duck plague virus |
| CN109234239B (en) * | 2018-07-13 | 2021-09-14 | 广东永顺生物制药股份有限公司 | Method for culturing duck plague virus |
| CN109207439A (en) * | 2018-10-09 | 2019-01-15 | 华农（肇庆）生物产业技术研究院有限公司 | A kind of full suspension culture method of duck plague virus |
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