CN106176729B - Application of the Fenamidone in preparation prevention and treatment BmNPV infected silkworm cell drug - Google Patents
Application of the Fenamidone in preparation prevention and treatment BmNPV infected silkworm cell drug Download PDFInfo
- Publication number
- CN106176729B CN106176729B CN201610566459.7A CN201610566459A CN106176729B CN 106176729 B CN106176729 B CN 106176729B CN 201610566459 A CN201610566459 A CN 201610566459A CN 106176729 B CN106176729 B CN 106176729B
- Authority
- CN
- China
- Prior art keywords
- cell
- fenamidone
- bmnpv
- treatment
- application
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
- A61K31/4164—1,3-Diazoles
- A61K31/4166—1,3-Diazoles having oxo groups directly attached to the heterocyclic ring, e.g. phenytoin
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Application of the Fenamidone in preparation prevention and treatment BmNPV infected silkworm cell drug, belong to molecular biology and field of virology, when Fenamidone using final concentration more than 1 μ g/mL when can completely inhibit the duplication of BmNPV, and the duplication that can continue to inhibit BmNPV after BmNPV completes to the infecting of cell, has therapeutic effect.The present invention can be used for preventing and treating the medicament research and development and production of silkworm blood type pus illness, have application and popularization value.
Description
Technical field
The invention belongs to molecular biology and field of virology, are related to Fenamidone in preparation and prevent and treat BmNPV infected silkworm
Application in cell drug.
Background technique
Silkworm blood type pus illness is that a kind of disease the most serious, infectiousness pole are endangered in the big virosis of sericulture three
By force, it breeds silkworms and more generally occurs in producing in recent years, and generating unit divides the case where having no harvest in peasant household or certain sericulture region frequently.
The cause of disease of silkworm blood type pus illness be bombyx mori nuclear polyhydrosis virus (Bombyx mori nucleopolyhedrovirus,
BmNPV), the Tobamovirus Rhabdoviridae, Nucleopolyhedrovirus are a kind of with cyst membrane, double-stranded DNA virus, it is in disease
It will form two kinds of virion, i.e. inclusion body derived virus particle and plasmavirus particle in malicious reproduction process, the former is with food
Object enters Midgut of Silkworm, Bombyx Mori infection part midgut epithelial cells, and infected midgut epithelial cells can generate a large amount of plasmavirus particles, newborn bud
Raw virion infects other tissues in turn leads to cell cracking, and polypide is finally dead.Sick silkworm, pupa, the corpse of moth, pus and by
Silkworm faeces, rearing instrument, the silk cocoon of pollution there is strong infectiousness, be the main biography of nuclear polyhedrosis with a large amount of virus
Dye source.
Specific medicament there is no for the treatment of silkworm blood type pus illness at present, it is tight mainly to pass through the reinforcement sericulture phase in production
Lattice sterilize to prevent the infection of BmNPV, and disinfectant main component has chlorinated product and formaldehyde formulations etc..But silkworm blood type purulence
The incubation period of disease is short, rapid onset, and naked eyes are difficult to discover when infection, when naked eyes are found, then often in morbidity advanced stage or breaks out
Phase is then difficult effectively treat and then retrieve a loss once virus infection silkworm body, occurs disease, exploitation treatment silkworm blood
The drug of liquid type pus illness is silkworm raiser or the very urgent expectation of portion of techniques personnel.
Existing experimental study proof B- propiolactone, the chemical substances such as nalidixic acid morbidity tool metainfective to silkworm BmNPV
There is certain inhibitory effect, but its effect is still mainly reflected in prevention rather than treats.Fenamidone is that imidazolone type kills
Microbial inoculum belongs to mitochondrial respiratory inhibitor, has good protection and therapeutic activity to downy mildew caused by Oomycete fungal.Invention
People has found that Fenamidone has apparent inhibitory effect to BmNPV during screening silkworm blood type pus illness therapeutic compounds,
Fenamidone is applied to virus at present and BmNPV prevention and treatment aspect there is no patent and document report.
Summary of the invention
The technical issues of solution: the present invention provides a kind of Fenamidone and prevents and treats BmNPV infected silkworm cell drug in preparation
In application, another object, which is to provide, to be used to prevent and treat the suitable concentration of BmNPV infected silkworm cell using Fenamidone.
Technical solution: application of the Fenamidone in preparation prevention and treatment BmNPV infected silkworm cell drug.
The effective concentration of Fenamidone is 1 μ g/mL.
BmNPV infected silkworm cell drug is prevented and treated, effective component is the Fenamidone being dissolved in DMSO, and concentration is 1 μ g/
mL。
Specific control method are as follows:
1. prevention and treatment of the Fenamidone to the BmNPV infection for carrying eGFP reporter gene
(1) Fenamidone is dissolved with DMSO, compound concentration is the storing liquid of 10mg/mL.
(2) about 10 are inoculated in 35mm Tissue Culture Dish respectively5The bombyx mori cell of a/ware, after cell is adherent, with preparing
Fenamidone storing liquid be added cell in, making final concentration is respectively 0.01-1 μ g/mL, be incubated for 10-120min, incubated cell temperature
Degree is 20-29 DEG C, using isometric DMSO Incubating Solution as control.After incubation, with MOI=10 Carrying Green Fluorescent Protein gene
BmNPV virus infected cell, 27 DEG C after culture 72 hours in fluorescence microscopy microscopic observation cell, and virus titer is surveyed
It is fixed.
(3) about 10 are inoculated in 35mm Tissue Culture Dish respectively5The bombyx mori cell of a/ware after cell is adherent, uses MOI=
1 μ g/mL miaow is added behind 6,12,24 hours after infection in the BmNPV virus infected cell of 10 Carrying Green Fluorescent Protein genes
Cycloheximide triazole in fluorescence microscopy microscopic observation cell after 72 hours, and is measured virus titer.
The utility model has the advantages that the present invention provides one kind using Fenamidone as principle active component, preparation prevents and treats silkworm blood type purulence
The drug of disease.When Fenamidone using final concentration more than 1 μ g/mL when can completely inhibit the duplication of BmNPV, and
BmNPV completes have therapeutic effect to the duplication that can continue to inhibit BmNPV after the infecting of cell.The present invention can be used for preventing and treating
The medicament research and development and production of silkworm blood type pus illness have application and popularization value.
Detailed description of the invention
The BmNPV recombinant virus BmBac-egfp schematic diagram of Fig. 1, Carrying Green Fluorescent Protein gene.
Fig. 2, cell restrovirus infection cell fluorogram is handled using various concentration Fenamidone.
Fig. 3, cell restrovirus titre is handled using various concentration Fenamidone.
Different time points are added Fenamidone and handle cell fluorescence figure after Fig. 4, infection.
Specific embodiment
Embodiment 1
1. carrying the building of the BmNPV of egfp reporter gene
Egfp segment is cloned into BamHI (Takara) and the position XhoI (Takara) of pFastBac1 (Invitrogen)
Point constructs donor plasmid pFastBac-egfp, obtains BmBac-egfp (Fig. 1) by swivel base, BmBac-egfp DNA is transfected
After BmN (silkworm gonad cell) cell, green fluorescence expression can be observed under fluorescence microscope.Its cell conditioned medium is used to feel
BmN cell is contaminated, harvests virus after infection 72 hours, measures virus titer with Endpoint Dilution Method, virus-4 DEG C refrigerator is kept in dark place.
2. the inoculation of cell and the storing liquid of Fenamidone are prepared
(1) about 10 are respectively inoculated in the Tissue Culture Dish of 35mm (Corning) respectively5It is closed in each cell ware of BmN cell
The TC100 insect cell medium of the 10%FBS of 2mL.
(2) 27 DEG C of culture about 12h-24h are used for following experiment after cell is adherent.
(3) storing liquid of Fenamidone is prepared
The Fenamidone storing liquid for being 10mg/mL with DMSO compound concentration.
3. working as the final concentration of 0 μ g/mL of Fenamidone, i.e. cellular control unit, the BmNPV of egfp reporter gene is carried to thin
Born of the same parents' efficiency of infection
(1) each inoculation 10 is taken53 culture dishes of cell close the TC100 insect of the 10%FBS of 2mL in each cell ware
Cell culture medium.Old culture medium is removed, the TC100 culture medium of 2 μ L DMSO Yu 2mL 10%FBS, 27 DEG C of cultures are separately added into
The BmBac-egfp virus of MOI=10 is added in 30min.
(2) after 72h, 3 wares are placed in fluorescence inverted microscope, observation egfp expression situation is shown in Fig. 2
(DMSO)。
(3) 10 μ L cell culture fluids are taken, measure virus titer with Endpoint Dilution Method.
As the result is shown when 0 μ g/mL Fenamidone processing cell, the cell of BmNPV infection generates a large amount of fluorescence, virus titer
Log10It (TCID50) is 9.87 (see Fig. 3 (DMSO)).
Embodiment 2
When the final concentration of 1 μ g/mL of Fenamidone, to carry egfp reporter gene BmNPV infection prevention and treatment:
(1) each inoculation 10 is taken53 culture dishes of cell close the TC100 insect of the 10%FBS of 2mL in each cell ware
Cell culture medium.Old culture medium is removed, is separately added into the Fenamidone storing liquid of 2 μ L 10mg/mL with 2mL 10%FBS's
The BmBac-egfp virus of MOI=10 is then added in TC100 culture medium, 27 DEG C of culture 30min.
(2) after 72h, 3 wares are placed in fluorescence inverted microscope, observation egfp expression situation is shown in Fig. 2
(T1)。
(3) 10 μ L cell culture fluids are taken, measure virus titer with Endpoint Dilution Method.
As the result is shown when 1 μ g/mL Fenamidone processing cell, the cell of BmNPV infection does not have fluorescence generation, has reached complete
Complete to inhibit, virus titer is 0 (Fig. 3 T1).
Embodiment 3
When the final concentration of 0.25 μ g/mL of Fenamidone, to carry egfp reporter gene BmNPV infection prevention and treatment:
(1) each inoculation 10 is taken53 culture dishes of cell close the TC100 insect of the 10%FBS of 2mL in each cell ware
Cell culture medium.Old culture medium is removed, the Fenamidone storing liquid and 2mL 10%FBS of 0.5 μ L 10mg/mL are separately added into
TC100 culture medium, 27 DEG C of culture 30min, then be added MOI=10 BmBac-egfp virus.
(2) after 72h, 3 wares are placed in fluorescence inverted microscope, observation egfp expression situation is shown in Fig. 2
(T0.25)。
(3) 10 μ L cell culture fluids are taken, measure virus titer with Endpoint Dilution Method.
As the result is shown when 0.25 μ g/mL Fenamidone processing cell, the fluorescence of BmNPV infection cell is obvious compared with the control
It reduces, virus titer Log10It (TCID50) is 8 (Fig. 3 T0.25).
Embodiment 4
When the final concentration of 0.1 μ g/mL of Fenamidone, to carry egfp reporter gene BmNPV infection prevention and treatment:
(1) each inoculation 10 is taken53 culture dishes of cell close the TC100 insect of the 10%FBS of 2mL in each cell ware
Cell culture medium.Old culture medium is removed, the Fenamidone storing liquid and 2mL 10%FBS of 0.2 μ L 10mg/mL are separately added into
TC100 culture medium, 27 DEG C of culture 30min, then be added MOI=10 BmBac-egfp virus.
(2) after 72h, 3 wares are placed in fluorescence inverted microscope, observation egfp expression situation is shown in Fig. 2
(T0.1)。
(3) 10 μ L cell culture fluids are taken, measure virus titer with Endpoint Dilution Method.
As the result is shown when 0.1 μ g/mL Fenamidone processing cell, the fluorescence of BmNPV infection cell is obvious compared with the control
It reduces, but more than the cell of 0.25 μ g/mL Fenamidone processing, virus titer Log10It (TCID50) is 9.13 (Fig. 3 T0.1).
Embodiment 5
When the final concentration of 0.01 μ g/mL of Fenamidone, to carry egfp reporter gene BmNPV infection prevention and treatment:
(1) each inoculation 10 is taken53 culture dishes of cell close the TC100 insect of the 10%FBS of 2mL in each cell ware
Cell culture medium.Old culture medium is removed, the Fenamidone storing liquid and 2mL 10%FBS of 0.02 μ L 10mg/mL are separately added into
TC100 culture medium, 27 DEG C of culture 30min, then be added MOI=10 BmBac-egfp virus.
(2) after 72h, 3 wares are placed in fluorescence inverted microscope, observation egfp expression situation is shown in Fig. 2
(T0.01)。
(3) 10 μ L cell culture fluids are taken, measure virus titer with Endpoint Dilution Method.
As the result is shown when 0.1 μ g/mL Fenamidone processing cell, the fluorescence of BmNPV infection cell difference compared with the control
It is unobvious, virus titer Log10(TCID50) it is 9.65 (Fig. 3 T0.01), shows that Fenamidone does not have BmNPV under the concentration
Control efficiency.
Embodiment 6
Fenamidone is added when the final concentration of 1 μ g/mL of Fenamidone, after infection to carrying egfp reporter gene
The prevention and treatment of BmNPV infection:
(1) each inoculation 10 is taken53 culture dishes of cell close the TC100 insect of the 10%FBS of 2mL in each cell ware
Cell culture medium.Old culture medium is removed, the BmBac-egfp virus of MOI=10 is then added；The setting time is infection at this time
The Fenamidone storing liquid of 2 μ L 10mg/mL, 27 DEG C of cultures are added after 0 hour afterwards, 6,12,24 hours.
After (2) 72 hours, 3 wares are placed in fluorescence inverted microscope, observation egfp expression situation is shown in figure
4。
BmNPV completes also to have BmNPV to 1 μ g/mL Fenamidone processing cell is added after the infection of cell as the result is shown
Complete inhibitory effect indicates that 1 μ g/mL Fenamidone can carry out virus sweep and treatment to BmNPV infection cell.
Finally it should be noted that when Fenamidone concentration described in embodiment described above and embodiment is with handling
Between being merely to illustrate property purpose rather than limit, it should be appreciated by those of ordinary skill in the art that in cell state or cell
The small difference of culture medium physicochemical property such as pH value etc. can in the form and details make it various
Change, without departing from the spirit and scope of the present invention defined by the appended claims, and is included in spirit herein
With within the scope of.
Claims (2)
1. application of the Fenamidone in preparation prevention and treatment BmNPV infected silkworm cell drug.
2. application according to claim 1, it is characterised in that the effective concentration of Fenamidone is 1 μ g/mL.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610566459.7A CN106176729B (en) | 2016-07-18 | 2016-07-18 | Application of the Fenamidone in preparation prevention and treatment BmNPV infected silkworm cell drug |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610566459.7A CN106176729B (en) | 2016-07-18 | 2016-07-18 | Application of the Fenamidone in preparation prevention and treatment BmNPV infected silkworm cell drug |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106176729A CN106176729A (en) | 2016-12-07 |
| CN106176729B true CN106176729B (en) | 2018-12-14 |
Family
ID=57494062
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610566459.7A Active CN106176729B (en) | 2016-07-18 | 2016-07-18 | Application of the Fenamidone in preparation prevention and treatment BmNPV infected silkworm cell drug |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106176729B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116196320B (en) * | 2023-02-13 | 2024-09-17 | 江苏科技大学 | Application of plantain D in inhibiting microsporidian and preparing medicine for preventing or treating corpuscle disease |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1023832A1 (en) * | 1999-01-29 | 2000-08-02 | American Cyanamid Company | Aqueous suspension concentrate |
| CN104370891A (en) * | 2014-10-17 | 2015-02-25 | 中国农业大学 | 5-(butane lactone-3-ethylidene)-2-amino imidazolinone compounds, preparation method and application thereof |
-
2016
- 2016-07-18 CN CN201610566459.7A patent/CN106176729B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1023832A1 (en) * | 1999-01-29 | 2000-08-02 | American Cyanamid Company | Aqueous suspension concentrate |
| CN104370891A (en) * | 2014-10-17 | 2015-02-25 | 中国农业大学 | 5-(butane lactone-3-ethylidene)-2-amino imidazolinone compounds, preparation method and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| Kathryn Homa等.Evaluation of Fungicides for the Control of Peronospora belbahrii on Sweet Basil in New Jersey.《Plant Disease》.2014,第98卷(第11期),第1561-1566页. * |
| 肖春芳等.钙％霜霉威·咪唑菌酮悬浮剂对马铃薯晚疫病的防治效果.《植物保护》.2014,第40卷(第2期),第171-174页. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106176729A (en) | 2016-12-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| McNeil et al. | Contributions of immune responses to developmental resistance in Lymantria dispar challenged with baculovirus | |
| US11591575B2 (en) | Compositions and methods for pest control management | |
| CN104027347B (en) | The application of first group-beta-cyclodextrin in preparation control BmNPV infected silkworm cell drug | |
| Geisler et al. | Adventitious viruses in insect cell lines used for recombinant protein expression | |
| JP2020178698A (en) | Expression of recombinant proteins in trichoplusia ni pupae | |
| Rohrmann | The baculovirus replication cycle: Effects on cells and insects | |
| CN102220326B (en) | SiRNA of SjLGL gene of schistosoma japonica and use thereof | |
| GB2463783A (en) | A method for preparing antigens of foot-and-mouth disease virus | |
| CN1696294A (en) | High-performance bioreactor for baculovirus of insects | |
| CN106957825A (en) | A kind of rice leaf spot bacteria bacteriophage of separation and its application | |
| CN106176729B (en) | Application of the Fenamidone in preparation prevention and treatment BmNPV infected silkworm cell drug | |
| Jiang et al. | Viricidal activity of several disinfectants against African swine fever virus | |
| Hoover et al. | Systemic component to intrastadial developmental resistance in Lymantria dispar to its baculovirus | |
| CN109364053A (en) | A kind of small molecule compound is preparing the application in anti-silkworm baculovirus infection medicine | |
| Rabalski et al. | An alphabaculovirus isolated from dead Lymantria dispar larvae shows high genetic similarity to baculovirus previously isolated from Lymantria monacha–an example of adaptation to a new host | |
| CN103555653B (en) | In-vitro construction method and application of fugu rubripes ovarian cell line | |
| CN109223784B (en) | Application of small molecular compound in preparation of silkworm antiviral drugs | |
| CN103898112B (en) | The recombinant baculovirus of the dsRNA of expression inhibiting corpus allatum hormone methyl transferase gene | |
| CN103667173B (en) | A kind of primary culture method of brown paddy plant hopper cell | |
| Mukawa et al. | Nucleopolyhedrovirus enhancement by a fluorescent brightener in Mythimna separata (Lepidoptera: Noctuidae) | |
| Chakraborty et al. | In vivo enhancement of nucleopolyhedrovirus of oriental armyworm, Mythimna separata using spindles from Helicoverpa armigera entomopoxvirus | |
| TW200846471A (en) | Lymantria xylina cell strain | |
| CN106318900A (en) | Method for rapidly preparing large number of chick embryo fibroblasts | |
| CN106047822B (en) | Methyl-B-cyclodextrin is expanding the application in bombyx mori nuclear polyhydrosis virus host domain | |
| US7955793B2 (en) | Cultured silkworm cells capable of highly efficient baculovirus production and protein production |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| EE01 | Entry into force of recordation of patent licensing contract | ||
| EE01 | Entry into force of recordation of patent licensing contract |
Application publication date: 20161207 Assignee: Center for technology transfer Jiangsu University of Science and Technology Assignor: JIANGSU University OF SCIENCE AND TECHNOLOGY Contract record no.: X2021980006173 Denomination of invention: Application of imidazolidone in preparation of drugs for prevention and treatment of BmNPV infection of silkworm cells Granted publication date: 20181214 License type: Common License Record date: 20210714 |
|
| EC01 | Cancellation of recordation of patent licensing contract | ||
| EC01 | Cancellation of recordation of patent licensing contract |
Assignee: Center for technology transfer Jiangsu University of Science and Technology Assignor: JIANGSU University OF SCIENCE AND TECHNOLOGY Contract record no.: X2021980006173 Date of cancellation: 20210826 |