CN106047822B - Methyl-B-cyclodextrin is expanding the application in bombyx mori nuclear polyhydrosis virus host domain - Google Patents
Methyl-B-cyclodextrin is expanding the application in bombyx mori nuclear polyhydrosis virus host domain Download PDFInfo
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Abstract
Methyl-B-cyclodextrin is expanding the application in bombyx mori nuclear polyhydrosis virus host domain, after containing a certain concentration M β CD processing BmNPV non-host cell, BmNPV infection non-host insects cell efficiency can significantly be improved, make BmNPV that can replicate amplification in non-host cell, generate the virion and polyhedral body of infectious, and control cell BmNPV then not reproducible completely, polyhedrosis generation is not observed more.The present invention solves the problems, such as that BmNPV host domain is narrow, and BmNPV can be made to replicate in non-host cell, and efficient infection non-host cell generates polyhedral body.The disadvantages of baculoviral is narrow as biological insecticides host range, and desinsection speed is slow can also be improved simultaneously, may additionally facilitate the development of the researchs such as the mechanism of insect baculovirus and host cell interaction, baculoviral receptor and baculoviral invasion host.
Description
Technical field
The invention belongs to biological insecticides and baculoviral to learn field, be related to methyl-B-cyclodextrin (Methyl- β-
Cyclodextrin, abbreviation M β CD are also expressed as MBCD, Me- β CD, MeBCD) expanding bombyx mori nuclear polyhydrosis virus place
Application in main domain.
Background technique
Baculoviral is the maximum monoid in known insect viruses, be discovery earliest, most study and Practical significance very
Big insect viruses, are a kind of togavirus of closed hoop double-stranded DNA, and Genome Size is about 80~180kb.Due to rod-shaped
Virus is the controlling elements of agriculture and forestry primary pest again, therefore is also widely used for biological insecticides.
Baculoviral is mostly isolated from insect, all with 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 genomes have very high homology, the host domain ratio BmNPV wide of AcMNPV is obtained
More, AcMNPV can be replicated in sf9, sf21, TN-368, CLS-79 cell line, but cannot be replicated 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.
Cannot infect (Beijing Lv Hongsheng insect viruses molecular biology [M]: Scientia Agricultura Sinica publishing house, 1998, p170-
174.)。
It is existing at present that Baculovirus Gene group much is transformed using molecular biology and genetic engineering means, expand rod-shaped disease
(Guo Rui etc. can infect the building of the recombinant bombyx mori nuclear polyhedrosis virus of tea geometrid, Chinese agriculture section to the report of malicious host domain
Learn, 2012,45 (16): 3288-3296), but yet there are no relevant report in terms of expanding baculoviral host domain using M β CD.
Silkworm is the important economic insects in China, and pus illness caused by BmNPV is most serious disease in Sericultural production,
BmNPV infectiousness is very strong, but due to continuously breeding silkworms, causes virus to be killed not exclusively, every year since BmNPV infection leads to pus illness
Generation be up to Sericultural production total value 16% to Silk Industry bring massive losses.Silkworm increases rapidly in larval phase body length and weight
Add, to 5 ages, weight every, up to 5-7 grams, since silkworm volume is big, can produce a large amount of when BmNPV burst occurs
BmNPV virus, these viruses have to pass through stringent dissipation processing in production at present, propagate again to avoid virus.But such as
Fruit can be used the unavoidable hazardous material of this sericulture, so that it may turn waste into wealth.
Summary of the invention
The technical issues of solution: the present invention provides a kind of methyl-B-cyclodextrin and is expanding bombyx mori nuclear polyhydrosis virus place
Application in main domain, applicant's early-stage study find that when M β CD concentration is higher, M β CD can completely inhibit BmNPV invasion silkworm
BmN cell, but when concentration is lower, BmNPV invasion non-host cell, such as sf9 and sf21 but can be enhanced in it.I.e. with containing
After the cell culture fluid of a certain concentration M β CD is incubated for the non-host insects cell of BmNPV, then the cell handled with BmNPV infection,
BmNPV is remarkably improved to the efficiency of infection of non-host insects cell, this method is simple and clear, and it is low in cost convenient for operation,
It is easy to spread, it improves efficiency significant.
Technical solution: methyl-B-cyclodextrin is expanding the application in bombyx mori nuclear polyhydrosis virus host domain.
The working concentration of the methyl-B-cyclodextrin is 0.125-2mM.
The host is sf9, sf21, high five (Hi5), TN369, HzAM1.
Expand the composition of bombyx mori nuclear polyhydrosis virus host domain, effective component includes methyl-B-cyclodextrin.
The configuration of 1.M β CD (being purchased from Sigma company) solution and virus prepare
With 1 × PBS [being purchased from Sangon Biotech (Shanghai) Co., Ltd.] dissolution M β CD, compound concentration is 50mM's
Storing liquid.
BmBacJS13 is one plant of Bacmid [Huang JS et with BmNPV with 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 counting
Efficiency of infection of the present invention, by the polyhedrosis gene of reporter gene egfp and BmNPV under hsp70 manipulation
(polyhedra, polh) simultaneously swivel base arrive BmBacJS13 Tn7 swivel base insertion point, after recombination bacmid name BmBac-
Egfp-ph extracts the DNA of recombination bacmid, transfects bombyx mori cell, observes strong green fluorescence under fluorescence microscope, receives
After obtaining budding viral (Budded Virus, BV), continues on for infected silkworm cell and carries out virus amplification, harvest virus after 72h,
Virus titer is measured using Endpoint Dilution Method, 4 DEG C are kept in dark place, in all methods of the present invention.
2. various concentration M β CD incubated cell and the observation of the cell fluorescence of subsequent virus infection
The BmNPV non-host insects cell normally cultivated, such as sf9, sf21 etc., patch are inoculated in Tissue Culture Dish respectively
Wall for 24 hours after, be added in cell culture medium with prepared M β CD storing liquid, making M β CD final concentration is respectively 0.125-2mM, is incubated
20-60min is educated, incubated cell temperature is 24-29 DEG C, using 0mM M β CD (i.e. isometric 1 × PBS) as control.Incubation time
After arriving, the M β CD Incubating Solution of 1 × PBS and various concentration are removed respectively, it is thin with the TC-100 culture medium or other insects of serum-free
Born of the same parents' culture medium gently rinses cell 2 times (can not also wash), then uses 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 of infection 1h, is rinsed 2 times with serum-free insect cell culture medium
(can not also wash), is placed in 27 DEG C of incubator cultures, different disposal can be observed under fluorescence microscope in 6-72h after culture
Express the difference of fluorecyte quantity.Phase after infection can see virus with the cell that M β CD was incubated under an optical microscope
Polyhedrosis formation, and the cell of 1 × PBS incubation is compareed without polyhedrosis formation.Fig. 1 is that BmBac-egfp-ph infects non-place
Main insect cell sf21 is exemplary, and as a result, when being incubated for sf21 cell with 1 × PBS control, virus infection efficiency is very low, infection
72h does not have polyhedrosis formation afterwards, but with after the M β CD of 0.25mM processing, efficiency of infection is significantly improved, and cell is complete after infecting 72h
There is green fluorescence expression in portion, shows that BmNPV can be very good to replicate in sf21 cell, and can see polyhedrosis formation
(shown in red arrow).
3. various concentration M β CD incubated cell influences BmBac-egfp-ph infection rate
Be inoculated with the BmNPV non-host insects cell normally cultivated in 24 porocyte culture plates respectively, it is adherent for 24 hours after, use
Prepared M β CD storing liquid is added in cell culture fluid, and making M β CD final concentration is respectively 0.125~2mM, is incubated for 20-
60min, incubated cell temperature are 24-29 DEG C, using 0mM M β CD (i.e. isometric 1 × PBS) Incubating Solution as control.Incubation time
After arriving, the M β CD Incubating Solution of 1 × PBS and various concentration are removed respectively, is gently rushed with serum-free insect cell culture medium or PBS
It washes cell 2 times, is then infected with the BmBac-egfp-ph virus of MOI=10 Carrying Green Fluorescent Protein gene and do not existed together respectively
The cell of reason after 27 DEG C of infection 1h, removes unadsorbed virion and culture medium, with the insect cell medium of serum-free or
Person PBS is rinsed 2 times, and normal cell culture medium is added, and after being placed in 27 DEG C of incubator culture 6h, is removed cell culture medium, is used PBS
Suspension cell has the cell number of luciferase expression with flow cytometer statistics, utilizes t test Analysis various concentration M β CD incubated cell
With the difference in terms of virus feels efficiency compareed.Experiment sets three repetitions.Statistical result showed 0.125mM M β CD processing is thin
When born of the same parents, BmNPV infection cell efficiency about compare 2.5 times, and 0.25,0.5,0.75,1,1.5, treated by 2mM M β CD
Efficiency of infection is respectively to compare 5.5,5.6,6,6.3,6.7,5.9 times.
4.BmNPV viral growth curves measurement in the cell after M β CD incubation
The BmNPV non-host insects cell normally cultivated is inoculated in 6 porocyte culture plates respectively, after adherent, with preparation
Good M β CD storing liquid is added in cell culture fluid, makes the M β final concentration of 0.25mM of CD, 27 DEG C of incubation 30min, with 0mM M β
CD (i.e. isometric 1 × PBS) Incubating Solution is as control.After incubation time arrives, the M β CD of 1 × PBS and various concentration are removed respectively
Incubating Solution is gently rinsed cell 2 times 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 infection liquid after 27 DEG C of infection 2h, with the Insect cellculture of serum-free
Base rinses 2 times (being set as 0h with this time), and 27 DEG C of routine cultures of 2mL normal incubation medium are added, respectively after infection 0,12,
24,48,72,96h takes 10 μ L of Supernatant samples, using BmN cell, is dripped using the virus of Endpoint Dilution Method measurement each time point
Degree, experiment set and repeat three times, draw the one step growth curve of virus, to determine whether BmNPV virus can be in sf21 cell
Normal replication amplification.Fig. 3 is the viral one step growth curve of the final concentration of 0.25mM of M β CD and control PBS, can be sent out from figure
It is existing, it is straight line to the growth curve for impinging upon 0-96h, cell does not generate virion, shows after BmNPV infects sf21
BmNPV is in untreated non-host cell sf21 not reproducible, and after M β CD processing cell, 0 with 12h time point without virus
Particle generates, but late viral number of particles is begun to ramp up, and is continued until 96h after infection, shows that BmNPV infects restrovirus
It can replicate, and be generated with infective virion in non-host cell.
The utility model has the advantages that present invention firstly discovers that a kind of new application of M β CD.With contain a certain concentration M β CD medium treatment
After BmNPV non-host cell, BmNPV infection non-host insects cell efficiency can be significantly improved, makes BmNPV can be non-sensitive thin
Amplification is replicated in born of the same parents, generates the virion and polyhedral body of infectious, and control cell BmNPV then not reproducible completely, more
Polyhedrosis generation is not observed.The invention of the purposes solves the problems, such as that BmNPV host domain is narrow, BmNPV can be made in non-place
Chief cell duplication, efficient infection non-host cell generate polyhedral body.Baculoviral can also be improved as biological insecticides place simultaneously
The disadvantages of main narrow range, desinsection speed is slow, may additionally facilitate insect baculovirus and host cell interaction, baculoviral receptor
And the development of the researchs such as mechanism of baculoviral invasion host.Method provided by the invention is simple and clear, should be readily appreciated that, operates
It is simple and easy, it is high-efficient.
Detailed description of the invention
Fig. 1 is to handle sf21 cell using the final concentration of 0.25mM M β CD of heretofore described method (to compare at PBS
Reason), fluorescence and visible light compare figure after viral BmBac-egfp-ph infection 72h:
A and B is the fluorescence picture and visible light picture that BmBac-egfp-ph infects that PBS handles cell；C and D is BmBac-
Egfp-ph infects the fluorescence picture and visible light picture of cell after M β CD processing；Arrow show BmNPV polyhedral body in D.Amplification
Multiple be 640 ×.Control cell amount of fluorescence is considerably less as the result is shown, and treated that the nearly all cell of cell is felt
Dye, and the cell that M β CD was incubated for is not it is observed that polyhedral body, control treatment observe polyhedral body then.
Fig. 2 is after handling sf21 cell using the seven kinds of various concentration M β CD processing of heretofore described method and control PBS
BmNPV efficiency of infection compares:
BmNPV control is that BmBac-egfp-ph invades the sf21 cell infection efficiency handled without M β CD, AcMNPV control
For Autographa nuclear polyhedrosis virus (Autographa californica nucleopolyhedrovirus, AcMNPV)
To the efficiency of infection (MOI=1) of sf21, * * indicate each M β CD processing group compareed with BmNPV with significant difference (P <
0.001)。
Data are reproducible results three times in figure.After various concentration M β CD processing, cell infection rate, which is noticeably greater than, to be compareed, and
Reach similar infectious effect (MOI=1) with AcMNPV, sf21 is AcMNPV host cell).
Fig. 3 is to handle and compare PBS using heretofore described method 0.25mM M β CD to handle sf21, BmBac-egfp-
Ph viral growth curves in sf21 cell compare:
Ordinate is viral median tissue infective dose (TCID50)Log10Logarithm, abscissa are time point after infection.It is right
According to not having infective virion to discharge in PBS processing cell, viral growth curves are straight line, show BmBac-
Egfp-ph is expanded in control without duplication, and M β CD processing cell starts to release the virion of infectious after 12h
Son, and it is continued until 96h after infection, show that BmNPV can be replicated effectively in cell after M β CD processing, be expanded.
Specific embodiment
By taking BmNPV infects non-host insects cell sf21 as an example, illustrate specific implementation step of the invention:
The configuration of M β CD (being purchased from Sigma company) solution and virus prepare
With 1 × PBS [being purchased from Sangon Biotech (Shanghai) Co., Ltd.] dissolution M β CD, compound concentration is 50mM's
Storing liquid.
BmBacJS13 is one plant of Bacmid [Huang JS et with BmNPV with 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 counting
Efficiency of infection of the present invention, the reporter gene egfp and polyhedrosis gene swivel base under hsp70 is manipulated are to BmBacJS13's
Tn7 swivel base insertion point names BmBac-egfp-ph, extracts DNA, transfects bombyx mori cell, and harvest sprouts after virus, continues to use
Strong green fluorescence is observed under infected silkworm cell, fluorescence microscope, and virus, Endpoint Dilution Method measurement are harvested after 72h
Virus titer, 4 DEG C are kept in dark place, in following present invention the method.
Embodiment 1: final concentration of 0.25mM M β CD is incubated for, the raising to BmNPV infection non-host cell efficiency
About 5 × 10 are respectively inoculated in two 35mm Tissue Culture Dish respectively5The sf21 cell of a/ware after adherent, takes preparation
Good M β CD storing liquid is added thereto in a ware cell culture fluid, makes M β CD final concentration be respectively 0.25mM, with 0mM M β
Another ware is added as control in CD (i.e. 1 × PBS of respective volume) Incubating Solution, then 27 DEG C of incubation 30min remove 1 respectively
× PBS and M β CD Incubating Solution is gently rinsed cell 2 times with the TC-100 culture medium of serum-free, then carries green with MOI=10
The BmBac-egfp-ph virus of fluorescence protein gene and polyhedrosis gene infects the cell of different disposal, 27 DEG C of infection 2h respectively
Afterwards, it is rinsed 2 times with the TC-100 culture medium of serum-free, the conventional TC-100 culture medium containing 10% fetal calf serum is added, places
After 27 DEG C of incubator culture 72h, the variation of fluorescence microscope virus infection efficiency.As shown in Figure 1, when being handled with PBS
Only have the expression that only a few cell has green fluorescent protein when cell, after virus infection, but after with M β CD processing, Ji Husuo
There is cell to be infected, has the expression of green fluorescent protein, and can see polyhedrosis formation, show the method for the present invention
It can be very good to improve BmNPV to the efficiency of infection of non-host cell.
Embodiment 2: respectively with final concentration of 0.125,0.25,0.5,0.75,1,1.5, after the M β CD processing of 2mM, streaming
The variation of cell instrument statistics virus infection efficiency
About 1 × 10 is respectively inoculated in 24 porocyte culture plates5The sf21 in a/hole, it is adherent for 24 hours, take prepared M β respectively
1.25 μ L of CD storing liquid, 2.5 μ L, 5 μ L, 7.5 μ L, 10 μ L, 15 μ L, 20 μ L are added in cell culture fluid that (cell culture fluid is total
Volume is 500 holes μ L/), make M β CD final concentration be respectively 0.125,0.25,0.5,0.75,1,1.5,2mM, in BmNPV control and
It is added 20 μ L 1 × PBS in AcMNPV control, 27 DEG C are incubated for the M β CD Incubating Solution that 1 × PBS and various concentration are removed after 30min,
Cell 2 times are gently rinsed with the TC-100 culture medium of serum-free, then with MOI=10 Carrying Green Fluorescent Protein gene and polygonal
The BmBac-egfp-ph virus of body gene infects 0.125 respectively, 0.25,0.5,0.75,1,1.5,2mMM β CD is incubated for and PBS is incubated
The cell educated, and compared with the AcMNPV of MOI=1 infection PBS incubated cell, after 27 DEG C of infection 1h, with the TC- of serum-free
100 culture mediums rinse 2 times, after being placed in 27 DEG C of incubator culture 6h, remove cell culture medium, PBS suspension cell, fluidic cell
Instrument counts the cell number for having luciferase expression, and after determining various concentration M β CD incubated cell, BmNPV infects the effect of non-host cell
Rate.Fig. 2 be BmNPV infection non-host insects cell sf21 it is exemplary as a result, statistics display 0.125mM M β CD processing cell when,
BmNPV infection cell efficiency about compare 2.5 times, and 0.25,0.5,0.75,1,1.5,2mM M β CD treated infection effect
Rate is respectively to compare 5.5,5.6,6,6.3,6.7,5.9 times, reaches extremely significant horizontal (P < 0.001).
When the final concentration of 0.25mM of embodiment 3:M β CD, BmNPV infects the measurement of restrovirus growth curve
About 5 × 10 are inoculated in 6 porocyte culture plates respectively5A/hole sf21 cell after adherent, takes prepared M β CD
10 μ L of storing liquid is added in the cell culture fluid of attached cell sf21, and making M β CD final concentration is respectively 0.25mM (total volume
2mL), 27 DEG C of incubation 30min, using 10 μ L1 × PBS Incubating Solutions as control.After incubation time arrives, 1 × PBS and M β is removed respectively
CD Incubating Solution is gently rinsed cell 2 times with 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 of infection 1h, with no blood
Clear TC-100 culture medium rinses 2 times, is added normal cell culture fluid 2mL (being set as 0h with this time), 27 DEG C of normal cultures,
Distinguish 0,12,24,48,72,96h 10 μ L viral supernatants of collection after infection, is measured often using BmN cell by Endpoint Dilution Method
The titre of a time point virus, infection and titer determination set and repeat three times.According to the virus titer of different time points, virus is drawn
One step growth curve.Fig. 3 the result shows that, control PBS processing cell after, BmNPV infection after, do not infected in 0-96h virus
Property virion generate, and BmNPV infection M β CD processing cell after, infectious virion is released since 12h, always
Continue to 96h after infecting, shows that BmNPV virus can be replicated in M β CD processing cell, be expanded, generate infectious virion
Son.
Finally it should be noted that: M β CD concentration described in embodiment described above and embodiment and processing the time only
It for explanatory purposes rather than limits, it should be appreciated by those of ordinary skill in the art that in cell state or cell culture
The small difference of base physicochemical property etc. (such as pH value etc.), can make various change to it in the form and details
Become, it is more comprising other silkworm caryogram without departing from the spirit and scope of the present invention defined by the appended claims, while also
Angle precursor virus non-host cell, the non-silkworm insect cell such as sf9, high five (Hi5), TN369, HzAM1 and the training that suspends
Feeding above-mentioned cell, and be included within spirit and scope.
Claims (1)
1.0.125-2mM methyl-B-cyclodextrin is expanding application of the bombyx mori nuclear polyhydrosis virus host domain into sf21, described
Host is sf21.
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| Title |
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| BmCPV、BmNPV入侵的分子机制初步研究;王四妹;《中国优秀硕士学位论文全文数据库，基础科学辑，苏州大学硕士学位论文》;20160115;摘要，59、78 |
| Entry of Bombyx mori nucleopolyhedrovirus into BmN cells by cholesterol-dependent macropinocytic endocytosis;Jinshan Huang等;《Biochemical and Biophysical Research Communications》;20141231;第453卷;166-171 |
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