CN106047822A - Application of methyl-beta-cyclodextrins to expansion of silkworm bombyx mori nuclear polyhedrosis virus host range - Google Patents
Application of methyl-beta-cyclodextrins to expansion of silkworm bombyx mori nuclear polyhedrosis virus host range Download PDFInfo
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Abstract
The invention discloses application of methyl-beta-cyclodextrins (MbetaCD) to expansion of a silkworm bombyx mori nuclear polyhedrosis virus (BmNPV) host range. After MbetaCD with a certain concentration is used for processing BmNPV non-host cells, the efficiency at which BmNPV infects non-host insect cells can be remarkably improved, BmNPV can be duplicated and amplified in the non-host cells, infectious virus particles and polyhedral bodies are produced, contrast cells BmNPV can not be duplicated at all, and no polyhedral bodies can be observed. The problem that the BmNPV host range is narrow is solved, and BmNPV can be duplicated in the non-host cells and efficiently infects the non-host cells to produce polyhedral bodies. Meanwhile, the defects that baculovirus is narrow in host range, low in insect killing speed and the like when serving as a biological insecticide can be overcome, and the development of research on the interaction of insect baculovirus and host cells, the mechanism of baculovirus receptors and baculovirus invading a host and the like can also be promoted.
Description
Technical field
The invention belongs to biological insecticides and baculovirus and learn field, relate to methyl-B-cyclodextrin (Methyl-β-
Cyclodextrin, is called for short M β CD, is also expressed as MBCD, Me-β CD, MeBCD) expanding bombyx mori nuclear polyhydrosis virus place
Application in main territory.
Background technology
Baculovirus is the maximum monoid in known insect viruses, be find the earliest, most study and Practical significance very
Big insect viruses, are the togavirus of a kind of closed hoop double-stranded DNA, and Genome Size is about 80～180kb.Due to shaft-like
Virus is again the controlling elements of agriculture and forestry primary pest, is therefore also widely used for biological insecticides.
Baculovirus is mostly isolated from insecticide, all has the host domain of relative narrowness.Autographa
Californica nuclear polyhedrosis virus (Autographa californica nucleopolyhedrovirus AcMNPV) and silkworm
Nuclear polyhedrosis virus (Bombyx mori nucleopolyhedrovirus, BmNPV) is most commonly used two kinds of research
Insect baculovirus, although two viral genome have the highest homology, but the host domain of AcMNPV is wider than BmNPV to be obtained
Many, AcMNPV can replicate in sf9, sf21, TN-368, CLS-79 cell line, but can not replicate in Bombyx noriN cell, and
BmNPV is then different from AcMNPV, only replicates in the cell lines such as BmN, Bm5, several to non-host cell sf9, sf21, TN-368 etc.
Can not infect (Lv Hongsheng. insect viruses molecular biology [M]. Beijing: Scientia Agricultura Sinica publishing house, 1998, p170-
174.)。
Have at present and much utilize molecular biology and genetic engineering means transformation Baculovirus Gene group, expand shaft-like disease
(Guo Rui etc. can infect the structure of the recombinant bombyx mori nuclear polyhedrosis virus of tea geometrid, Chinese agriculture section to the report of poison host domain
Learn, 2012,45 (16): 3288-3296), but utilize M β CD to expand baculovirus host domain aspect and yet there are no relevant report.
Silkworm is the economic insects that China is important, and the grasserie that BmNPV causes is disease the most serious in Sericultural production,
BmNPV infectiousness is very strong, but owing to breeding silkworms continuously, causes virus to kill not exclusively, causes grasserie owing to BmNPV infects every year
The massive losses occurring to bring to Silk Industry be up to Sericultural production total value 16%.Silkworm increases rapidly in larval phase body length and body weight
Adding, to 5 ages, body weight every, up to 5-7 gram, owing to silkworm volume is big, can produce substantial amounts of when BmNPV burst occurs
BmNPV virus, in producing at present, these viruses have to pass through strict dissipation process, to avoid virus again to propagate.But such as
The hazardous material that fruit can be not fee from this sericulture is used, it is possible to turn waste into wealth.
Summary of the invention
Solve the technical problem that: the present invention provides a kind of methyl-B-cyclodextrin expanding bombyx mori nuclear polyhydrosis virus place
Application in main territory, applicant's early-stage Study finds when M β CD concentration is higher, and M β CD can completely inhibit BmNPV and invade silkworm
BmN cell, but when concentration is relatively low, it but can strengthen BmNPV and invade non-host cell, such as sf9 and sf21.I.e. with containing
After the cell culture fluid of finite concentration M β CD hatches the non-host insects cell of BmNPV, then the cell processed with BmNPV infection,
I.e. being remarkably improved the BmNPV efficiency of infection to non-host insects cell, the method is simple and clear, it is simple to operation, with low cost,
Easy to spread, improve efficiency notable.
Technical scheme: methyl-B-cyclodextrin application in expanding bombyx mori nuclear polyhydrosis virus host domain.
The working concentration of described methyl-B-cyclodextrin is 0.125-2mM.
Described host is sf9, sf21, high five (Hi5), TN369, HzAM1.
Expanding the compositions of bombyx mori nuclear polyhydrosis virus host domain, effective ingredient includes methyl-B-cyclodextrin.
Configuration and the virus of 1.M β CD (purchased from Sigma company) solution prepare
Dissolving M β CD with 1 × PBS [purchased from Sangon Biotech (Shanghai) Co., Ltd.], compound concentration is 50mM's
Store liquid.
BmBacJS13 is Bacmid [the Huang JS et that a strain and BmNPV have identical infection characterization
al.Construction of the Bac-to-Bac System of Bombyx mori
Nucleopolyhedroviru.Virologica Sinica.2007,22 (3): 218-225].For the ease of observing and statistics
Efficiency of infection of the present invention, the polyhedrosis gene of reporter gene egfp and BmNPV under hsp70 is handled
(polyhedra, polh) swivel base simultaneously names BmBac-to the Tn7 swivel base insertion point of BmBacJS13, the bacmid after restructuring
Egfp-ph, extracts the DNA of restructuring bacmid, transfects bombyx mori cell, observes strong green fluorescence under fluorescence microscope, receives
After obtaining virus of sprouting (Budded Virus, BV), continue on for infected silkworm cell and carry out virus amplification, results virus after 72h,
Utilizing Endpoint Dilution Method to measure virus titer, 4 DEG C keep in Dark Place, in all methods of the present invention.
2. variable concentrations M β CD incubated cell and the cell fluorescence with restrovirus infection are observed
The normal BmNPV non-host insects cell cultivated of inoculation, such as sf9, sf21 etc., patch in Tissue Culture Dish respectively
After wall 24h, store liquid with the M β CD prepared and join in cell culture medium, make M β CD final concentration be respectively 0.125-2mM, incubate
Educating 20-60min, incubated cell temperature is 24-29 DEG C, using 0mM M β CD (i.e. equal-volume 1 × PBS) as comparison.Incubation time
After arriving, removing the M β CD Incubating Solution of 1 × PBS and variable concentrations respectively, TC-100 culture medium or other insecticide with serum-free are thin
Born of the same parents' culture medium rinses cell 2 times (also can not wash) gently, then with the BmBac-of MOI=5-50 Carrying Green Fluorescent Protein gene
Egfp-ph virus infects the cell of different disposal respectively, after 27 DEG C are infected 1h, rinses 2 times by serum-free insect cell culture medium
(also can not wash), is positioned over 27 DEG C of incubators and cultivates, and 6-72h after cultivation i.e. can be observed different disposal under fluorescence microscope
Express the difference of fluorecyte quantity.Phase after infection, the cell hatched with M β CD can see virus under an optical microscope
Polyhedrosis formation, and compare cell that 1 × PBS hatches without polyhedrosis formation.Fig. 1 is that BmBac-egfp-ph infects non-place
The result of main insect cell sf21 example, when hatching sf21 cell with 1 × PBS control, virus efficiency of infection is the lowest, infects
Rear 72h does not has polyhedrosis formation, but after processing with the M β CD of 0.25mM, efficiency of infection significantly improves, and after infecting 72h, cell is complete
Portion has green fluorescence to express, and shows that BmNPV can well replicate in sf21 cell, and can see polyhedrosis formation
(shown in red arrow).
3. BmBac-egfp-ph infection rate is affected by variable concentrations M β CD incubated cell
The normal BmNPV non-host insects cell cultivated of inoculation in 24 porocyte culture plates, after adherent 24h, uses respectively
The M β CD prepared stores liquid and joins in cell culture fluid, makes M β CD final concentration be respectively 0.125～2mM, hatches 20-
60min, incubated cell temperature is 24-29 DEG C, using 0mM M β CD (i.e. equal-volume 1 × PBS) Incubating Solution as comparison.Incubation time
After arriving, remove the M β CD Incubating Solution of 1 × PBS and variable concentrations respectively, rush gently by serum-free insect cell culture medium or PBS
Wash cell 2 times, then infect respectively by the BmBac-egfp-ph virus of MOI=10 Carrying Green Fluorescent Protein gene and do not exist together
The cell of reason, 27 DEG C are infected after 1h, remove unadsorbed virion and culture medium, with the insect cell medium of serum-free or
Person PBS rinses 2 times, adds normal cell culture medium, after being positioned over 27 DEG C of incubators cultivation 6h, removes cell culture medium, uses PBS
Suspension cell, has the cell number of luciferase expression, utilizes t test Analysis variable concentrations M β CD incubated cell by flow cytometer statistics
With the difference in terms of virus sense efficiency compareed.Experiment sets three repetitions.Statistical result showed 0.125mM M β CD processes thin
During born of the same parents, BmNPV infect cell efficiency be about comparison 2.5 times, and 0.25,0.5,0.75,1,1.5,2mM M β CD process after
Efficiency of infection is respectively comparison 5.5,5.6,6,6.3,6.7,5.9 times.
In 4.BmNPV cell after M β CD is hatched, viral growth curves measures
The normal BmNPV non-host insects cell cultivated of inoculation in 6 porocyte culture plates respectively, adherent after, with preparation
Good M β CD stores liquid and joins in cell culture fluid, makes the M final concentration of 0.25mM of β CD, hatches 30min for 27 DEG C, with 0mM M β
CD (i.e. equal-volume 1 × PBS) Incubating Solution is as comparison.After incubation time arrives, remove the M β CD of 1 × PBS and variable concentrations respectively
Incubating Solution, rinses cell 2 times gently with the insect cell medium of serum-free, then uses MOI=20BmBac-egfp-ph BV
Virus infects the cell of different disposal respectively, removes virus and infect liquid, with the Insect cellculture of serum-free after 27 DEG C of infection 2h
Base rinses 2 times (being set as 0h with this time), adds 27 DEG C of cellar cultures of 2mL normal incubation medium, respectively the most after infection 0,12,
24,48,72,96h take Supernatant samples 10 μ L, utilize BmN cell, the virus using Endpoint Dilution Method to measure each time point is dripped
Degree, experiment sets three repetition, draws the one step growth curve of virus, with determine BmNPV viral in sf21 cell the most permissible
Normal replication expands.Fig. 3 is the M final concentration of 0.25mM of β CD and the viral one step growth curve of comparison PBS, can send out from figure
Existing, after BmNPV infects sf21, it is straight line to the growth curve impinging upon 0-96h, cell does not produce virion, shows
BmNPV is at untreated non-host cell sf21 not reproducible, and after M β CD processes cell, does not has virus 0 with 12h time point
Particle produces, but late viral number of particles begins to ramp up, and is continued until 96h after infection, shows that BmNPV infects restrovirus
Can replicate in non-host cell, and generation has infective virion.
Beneficial effect: present invention firstly discovers that a kind of new application of M β CD.With containing finite concentration M β CD medium treatment
After BmNPV non-host cell, can significantly improve BmNPV infect non-host insects cell efficiency, make BmNPV can non-sensitive carefully
Born of the same parents replicate and expands, the virion of generation infectious and polyhedral body, compared with control cells BmNPV the most not reproducible, more
Do not observe polyhedrosis generation.The invention of this purposes, solves the problem that BmNPV host domain is narrow, and BmNPV can be made in non-place
Chief cell replicates, efficient infection non-host cell, produces polyhedral body.Also can improve baculovirus as biological insecticides place simultaneously
Main narrow range, the shortcomings such as parasite killing speed is slow, may additionally facilitate insect baculovirus and host cell interaction, baculovirus receptor
And the development that the mechanism etc. of baculovirus invasion host is studied.The method that the present invention provides is simple and clear, it is easy to understands, operate
Simple, efficiency is high.
Accompanying drawing explanation
Fig. 1 processes sf21 cell (at comparison PBS for utilizing heretofore described method final concentration of 0.25mM M β CD
Reason), fluorescence and visible ray comparison diagram after virus BmBac-egfp-ph infection 72h:
A Yu B is fluorescence picture and the visible ray picture that BmBac-egfp-ph infects PBS process cell；C Yu D is BmBac-
Egfp-ph infects fluorescence picture and the visible ray picture of cell after M β CD processes；In D, arrow show BmNPV polyhedral body.Amplify
Multiple is 640 ×.Result display compared with control cells amount of fluorescence is considerably less, and the nearly all cell of cell after processing all is felt
Dye, and the cell that M β CD was hatched is it is observed that polyhedral body, and control treatment does not then observe polyhedral body.
Fig. 2 is for utilizing seven kinds of variable concentrations M β CD of heretofore described method to process and after comparison PBS process sf21 cell
BmNPV efficiency of infection compares:
The sf21 cell infection efficiency that BmNPV comparison processes without M β CD for BmBac-egfp-ph invasion, AcMNPV compares
For Autographa nuclear polyhedrosis virus (Autographa californica nucleopolyhedrovirus, AcMNPV)
Efficiency of infection (MOI=1) to sf21, * * represent each M β CD process group compare with BmNPV have significant difference (P <
0.001)。
In figure, data are three reproducible results.After variable concentrations M β CD processes, cell infection rate is noticeably greater than comparison, and
Reaching close infectious effect (MOI=1) with AcMNPV, sf21 is AcMNPV host cell).
Fig. 3 is for utilizing heretofore described method 0.25mM M β CD to process and comparison PBS process sf21, BmBac-egfp-
Ph viral growth curves in sf21 cell compares:
Vertical coordinate is virus median tissue infective dose (TCID50)Log10Logarithm, abscissa is time point after infecting.Right
Processing in cell according to PBS does not has infective virion to discharge, and viral growth curves is straight line, shows BmBac-
Egfp-ph does not replicate amplification in comparison, and M β CD processes cell and starts to discharge the virion of infectious after 12h
Son, and it is continued until 96h after infection, show that BmNPV can effectively replicate in cell after M β CD processes, expand.
Detailed description of the invention
Infect as a example by non-host insects cell sf21 by BmNPV, the step that is embodied as of the present invention be described:
Configuration and the virus of M β CD (purchased from Sigma company) solution prepare
Dissolving M β CD with 1 × PBS [purchased from Sangon Biotech (Shanghai) Co., Ltd.], compound concentration is 50mM's
Store liquid.
BmBacJS13 is Bacmid [the Huang JS et that a strain and BmNPV have identical infection characterization
al.Construction of the Bac-to-Bac System of Bombyx mori
Nucleopolyhedroviru.Virologica Sinica.2007,22 (3): 218-225].For the ease of observing and statistics
Efficiency of infection of the present invention, by hsp70 handle under reporter gene egfp and polyhedrosis gene swivel base to BmBacJS13's
Tn7 swivel base insertion point, names BmBac-egfp-ph, extracts DNA, transfects bombyx mori cell, and results are sprouted after virus, continues to use
In infected silkworm cell, observing strong green fluorescence under fluorescence microscope, results virus after 72h, Endpoint Dilution Method measures
Virus titer, 4 DEG C keep in Dark Place, in method described in present invention below.
Embodiment 1: final concentration of 0.25mM M β CD is hatched, infects the raising of non-host cell efficiency to BmNPV
Each inoculation about 5 × 10 in two 35mm Tissue Culture Dishs respectively5The sf21 cell of individual/ware, adherent after, take preparation
Good M β CD stores liquid and is added thereto in a ware cell culture fluid, makes M β CD final concentration be respectively 0.25mM, with 0mM M β
CD (i.e. respective volume 1 × PBS) Incubating Solution addition another one ware, as comparison, is hatched 30min for 27 DEG C, is removed 1 the most respectively
× PBS and M β CD Incubating Solution, rinse cell gently 2 times by the TC-100 culture medium of serum-free, then carry green with MOI=10
The BmBac-egfp-ph virus of fluorescence protein gene and polyhedrosis gene infects the cell of different disposal respectively, and 27 DEG C are infected 2h
After, rinse 2 times by the TC-100 culture medium of serum-free, add the conventional TC-100 culture medium containing 10% hyclone, place
After 27 DEG C of incubators cultivate 72h, the change of fluorescence microscope virus efficiency of infection.As it is shown in figure 1, work as with PBS process
During cell, virus only has only a few cell after infecting have the expression of green fluorescent protein, but after processing with M β CD, Ji Husuo
There is cell to be all infected, have the expression of green fluorescent protein, and polyhedrosis formation can be seen, show the inventive method
The BmNPV efficiency of infection to non-host cell can well be improved.
Embodiment 2: respectively with final concentration of 0.125,0.25,0.5,0.75,1,1.5, after the M β CD of 2mM processes, streaming
The change of cell instrument statistics virus efficiency of infection
Each inoculation about 1 × 10 in 24 porocyte culture plates5The sf21 in individual/hole, adherent 24h, take the M β prepared respectively
CD stores liquid 1.25 μ L, 2.5 μ L, 5 μ L, 7.5 μ L, 10 μ L, 15 μ L, 20 μ L join in cell culture fluid that (cell culture fluid is total
Volume is 500 μ L/ holes), make M β CD final concentration be respectively 0.125,0.25,0.5,0.75,1,1.5,2mM, in BmNPV comparison and
AcMNPV comparison adds 20 μ L 1 × PBS, hatches removal 1 × PBS and the M β CD Incubating Solution of variable concentrations after 30min for 27 DEG C,
Cell is rinsed gently 2 times, then with MOI=10 Carrying Green Fluorescent Protein gene and polygonal by the TC-100 culture medium of serum-free
The BmBac-egfp-ph virus of body gene infects 0.125 respectively, 0.25,0.5,0.75,1,1.5,2mMM β CD hatches and incubates with PBS
The cell educated, and compare, after 27 DEG C are infected 1h, with the TC-of serum-free with the AcMNPV infection PBS incubated cell of MOI=1
100 culture medium rinse 2 times, after being positioned over 27 DEG C of incubators cultivation 6h, remove cell culture medium, PBS suspension cell, fluidic cell
Instrument statistics has the cell number of luciferase expression, and after determining variable concentrations M β CD incubated cell, BmNPV infects the effect of non-host cell
Rate.Fig. 2 is the result that BmNPV infects non-host insects cell sf21 example, when statistics display 0.125mM M β CD processes cell,
BmNPV infect cell efficiency be about comparison 2.5 times, and 0.25,0.5,0.75,1,1.5,2mM M β CD process after infection effect
Rate is respectively comparison 5.5,5.6,6,6.3,6.7,5.9 times, reaches pole significant level (P < 0.001).
During the final concentration of 0.25mM of embodiment 3:M β CD, BmNPV infects restrovirus growth curve and measures
About 5 × 10 are inoculated respectively in 6 porocyte culture plates5Individual/hole sf21 cell, adherent after, take the M β CD prepared
Storing in the cell culture fluid that liquid 10 μ L joins attached cell sf21, (cumulative volume is to make M β CD final concentration be respectively 0.25mM
2mL), hatch 30min for 27 DEG C, using 10 μ L1 × PBS Incubating Solution as comparison.After incubation time arrives, remove 1 × PBS and M β respectively
CD Incubating Solution, rinses cell gently 2 times by the TC-100 culture medium of serum-free, then uses MOI=20 Carrying Green Fluorescent Protein
The BmBac-egfp-ph virus of gene and polyhedrosis gene infects the cell of different disposal respectively, after 27 DEG C are infected 1h, uses depletion of blood
Clear TC-100 culture medium rinses 2 times, addition normal cell culture fluid 2mL (being set as 0h with this time), 27 DEG C of normal cultivations,
The most after infection 0,12,24,48,72,96h collect 10 μ L of supernatant viruses, utilize BmN cell to be measured by Endpoint Dilution Method every
The titre of individual time point virus, infects and titer determination sets three repetitions.According to the virus titer of different time points, draw virus
One step growth curve.Fig. 3 result shows, after comparison PBS processes cell, after BmNPV infects, does not infect in 0-96h virus
Property virion produce, and BmNPV infects M β CD and processes after cell, starts to discharge infectious virion from 12h, always
Last till 96h after infection, show that BmNPV virus can replicate in M β CD processes cell, expand, produce infectious virion
Son.
Finally should be noted that: the M β CD concentration described in embodiment described above and embodiment and the time of process are only
Unrestricted for explanatory purposes, it should be appreciated by those of ordinary skill in the art that cultivate at cell state or cell
The difference that base physicochemical property etc. (such as acid-base value etc.) are small, can make various changing to it in the form and details
Become, the spirit and scope of the present invention limited without departing from appended claims, also comprise other silkworm caryogram many simultaneously
Angle precursor virus non-host cell, such as sf9, high five (Hi5), the non-silkworm insect cell such as TN369, HzAM1 and the training that suspends
The above-mentioned cell supported, and within being included in spirit and scope.
Claims (4)
1. methyl-B-cyclodextrin application in expanding bombyx mori nuclear polyhydrosis virus host domain.
Application the most according to claim 1, it is characterised in that the working concentration of described methyl-B-cyclodextrin is 0.125-
2mM。
Application the most according to claim 1, it is characterised in that described host cell is sf9, sf21, high five
(Hi5)、TN369、HzAM1。
4. expand the compositions of bombyx mori nuclear polyhydrosis virus host domain, it is characterised in that effective ingredient includes that methyl-β-ring is stuck with paste
Essence.
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| CN112359064A (en) * | 2020-11-10 | 2021-02-12 | 江苏科技大学 | Application of methyl-beta-cyclodextrin in improving transduction efficiency of baculovirus to mammalian cells |
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| CN104027347A (en) * | 2014-05-26 | 2014-09-10 | 江苏科技大学 | Application of methyl-beta-cyclodextrin in preparation of medicines for preventing and controlling BmNPV from infecting silkworm cells |
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| CN104027347A (en) * | 2014-05-26 | 2014-09-10 | 江苏科技大学 | Application of methyl-beta-cyclodextrin in preparation of medicines for preventing and controlling BmNPV from infecting silkworm cells |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112359064A (en) * | 2020-11-10 | 2021-02-12 | 江苏科技大学 | Application of methyl-beta-cyclodextrin in improving transduction efficiency of baculovirus to mammalian cells |
| CN112359064B (en) * | 2020-11-10 | 2022-01-04 | 江苏科技大学 | Application of methyl-beta-cyclodextrin in improving transduction efficiency of baculovirus to mammalian cells |
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