CN105561340B - A kind of application of miRNA relevant to bombyx mori cell apoptosis in viral infection resisting - Google Patents
A kind of application of miRNA relevant to bombyx mori cell apoptosis in viral infection resisting Download PDFInfo
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Abstract
The invention discloses a kind of application of miRNA relevant to bombyx mori cell apoptosis in viral infection resisting.Present invention discover that bmo-miR-3378 expression quantity during silkworm BmN cell infection BmNPV significantly reduces, illustrate that it has specific function with host's interaction in virus；And be experimentally confirmed bmo-miR-3378 can by regulate and control BmN cell apoptosis, and then influence virus proliferation with infect, its new tiny RNA that can be used as regulation baculovirus infection, prevention and treatment antiviral to the silkworm in current sericultural production have important practical significance with breeding and promotion silkworm biological reactor industrialized development.
Description
Technical field
The present invention relates to technical field of cell biology, more particularly to a kind of miRNA relevant to bombyx mori cell apoptosis to exist
Application in viral infection resisting.
Background technique
Silkworm (Bombyx mori) is economic insects important in the world.China is the maximum silk cocoon in the whole world, silk production
State and Silk quality exported country (Gu Guoda etc., 2002).Sericulture industry occupies an important position in agricultural economy, is the weight of farmers' income
Want source.In addition, silkworm be used to express as bioreactor, produce foreign gene have its distinctive advantage (Zhang Yaozhou etc.,
2008).However, the generation of silkworm disease and the popular sustainable development for drastically influencing China's sericulture there.
Wherein, bombyx mori nuclear polyhydrosis virus (B.mori nuclear polyhedrosis virus, BmNPV, one kind
Baculoviral) caused by silkworm pus illness have in sericulture state, the world daily life of a family and break out as a kind of the most serious virosis is endangered, and pass
It contaminates strong, difficult to control, and easily causes huge economic loss.
Currently, the sericultures main producing region such as China Sichuan, Zhejiang, Jiangsu and Guangdong, causes silk cocoon to lose, about by BmNPV every year
Account for the 70~80% of silkworm disease total losses.For a long time, although in terms of the research of silkworm antiviral-mechanism and resistant variety
Oneself makes progress, but still has more problem not yet to solve (Lv Hongsheng, 2008).Moreover, also there is post for silkworm biological reactor
Main narrow range, specificity are high and foreign gene gene silencing may occur to the interaction of BmNPV regulatory mechanism in Silkworm, Bombyx mori
The problems such as phenomenon, serious restrict are developed using the biological farm of silkworm biological reactor industrialization development useful proteins.
Studies have shown that baculovirus infection can induce host insect Apoptosis (Apoptosis) (Pang Yi etc., 1998),
And Apoptosis is then considered as a kind of important mechanisms of insect defence virus infection, it is main to pass through the Suicide dead of host cell
Die with the infection of limiting virus and duplication (Schultz etc., 2009；Vandergaast etc., 2011).Exist in host cell
A variety of apoptosis regulating genes such as apoptosis inhibitory protein (Inhibitors of apoptosis proteins, IAPs)
(Verhagen etc., 2001) and API5 (Apoptosis inhibitor-5) (Morris etc., 2006) etc., adjustable ganglion cell is withered
The generation and termination died.In fact, IAPs is found in insect viruses earliest, host cell apoptosis can be prevented and realize disease
Poison duplication with proliferation (Clem etc., 2007；Sahdev etc., 2010).Therefore, in virus infection early stage, by promoting virus infection
Host cell occur apoptosis be one of effective viral resistant strategies.
Tiny RNA (microRNAs writes a Chinese character in simplified form miRNA or miR), which is a kind of length, has regulating and controlling effect in 18~25 bases
Non-coding RNA nucleic acid molecules.Tiny RNA plays an important role in the post-transcriptional control of gene, participate in insect growth, reproduction,
The various bioprocess such as nerve to occur, metabolism, immune and Apoptosis.
Studies have shown that tiny RNA can also participate in virus infection be immunoreacted regulation process (Fullaondo etc., 2012；
Hussain etc., 2014).Currently, 487 tiny RNA precursors and 563 mature tiny RNA (miRBase are found in silkworm body
V21,2014).Wherein, bmo-miR-3378 was most in by Cai Yi-Mei et al. using SOLiD sequencing technologies earlier than 2010
Identify and obtain in the full worm of silkworm and sericterium, predicted discovery has 43 potential target genes, relate generally to embryonic development, cell division,
Vesicle transport and neuroceptor and tumour generation, cause pathogeny imcrobe infection etc., but bmo-miR-3378 is in the intracorporal function of silkworm
Can not there be any report so far with mechanism of action.
Summary of the invention
Present invention discover that the bmo-miR-3378 in silkworm microRNAs can promote the apoptosis of bombyx mori cell, and pass through
The apoptosis of bombyx mori cell inhibits the proliferation of baculoviral in bombyx mori cell, to realize to the anti-of the silkworm of infection baculoviral
It controls.
Present invention finds bmo-miR-3378 to promote the application in bombyx mori cell apoptosis.
Specifically, the bombyx mori cell is Bombyx noriN cell.
Present invention finds bmo-miR-3378 to prepare the application in anti-silkworm baculovirus infection medicine.
Preferably, the baculoviral is nuclear polyhedrosis virus.
The present invention provides bmo-miR-3378 analogs to promote the application in bombyx mori cell apoptosis, which is characterized in that
The bmo-miR-3378 analog is double stranded RNA sequences, nucleotide sequence such as SEQ ID NO.9 and SEQ ID NO.10 institute
Show.
The present invention provides bmo-miR-3378 analogs to prepare the application in the drug that anti-silkworm baculovirus infects,
The bmo-miR-3378 analog is double stranded RNA sequences, nucleotide sequence such as SEQ ID NO.9 and SEQ ID NO.10 institute
Show.
The present invention provides bmo-miR-3378 mortifiers to inhibit the application in bombyx mori cell apoptosis, the bmo-miR-
The nucleotide sequence of 3378 mortifiers is as shown in SEQ ID NO.13.
The present invention also provides bmo-miR-3378 mortifiers to promote recombinant baculovirus to increase in silkworm biological reactor
The application grown and infected, the nucleotide sequence of the bmo-miR-3378 mortifier is as shown in SEQ ID NO.13.
The present invention also provides a kind of control methods of silkworm baculovirus disease, comprising: using bmo-miR-3378 or its
Analog transfects the bombyx mori cell by baculovirus infection；The nucleotide sequence of the bmo-miR-3378 such as SEQ ID NO.1
It is shown；The analog is double stranded RNA sequences, and nucleotide sequence is as shown in SEQ ID NO.9 and SEQ ID NO.10.
In order to verify above-mentioned discovery, the present invention is using the clear Bombyx noriN cell infection BmNPV of qRT-PCR method and transfection
Then Caspase activity is respectively adopted in the variation of bmo-miR-3378 expression quantity after bmo-miR-3378 analog and mortifier
Detection method, mtt assay, qRT-PCR method and Reed-Muench Endpoint Dilution Method have studied the variation of bmo-miR-3378 expression quantity to sense
Contaminate the influence of the Bombyx noriN cell apoptosis and vigor and BmNPV duplication and pathogenicity of BmNPV.The result shows that bmo-miR-
3378 can be by promoting BmN Apoptosis that BmNPV is inhibited to be proliferated and infect.
Particular content is as follows:
The present invention is examined using stem-loop method design, synthesis bmo-miR-3378 Specific PCR primers using qRT-PCR method
Survey the expression quantity variation of bmo-miR-3378 after comparing Bombyx noriN cell infection BmNPV.As a result, it has been found that in Bombyx noriN cell
12h and for 24 hours after BmNPV is infected, significant decrease (p < 0.05) occurs in bmo-miR-3378 expression quantity.
Then, inventor synthesizes bmo-miR-3378 analog and mortifier, transfects Bombyx noriN cell respectively, and use
The expression of qRT-PCR method detection bmo-miR-3378.As a result, it has been found that Bombyx noriN cell transfection bmo-miR-3378 is similar
After object, the extremely significant raising (p < 0.05) of bmo-miR-3378 expression quantity；And transfect the expression quantity after bmo-miR-3378 mortifier
Extremely significant reduction (p < 0.01).
On this basis, inventor to transfect respectively the Bombyx noriN cell of bmo-miR-3378 analog and mortifier into
Row virus infection processing, using the apoptosis induction situation of Caspase activity detection BmN cell.As a result, it has been found that through bmo-
MiR-3378 analog processing BmN cell connect poison for 24 hours within, Level of Apoptosis significantly increases (p < 0.05)；And it passes through
The BmN cell of bmo-miR-3378 mortifier processing is connecing within malicious 48h, and Level of Apoptosis significantly reduces (p < 0.05).This
Showing that bmo-miR-3378 expression quantity is raised and lowered significantly can induce respectively or inhibit host cell to the apoptotic response of virus.
Finally, present invention application bmo-miR-3378 analog and mortifier handle Bombyx noriN cell, then it is respectively adopted
Mtt assay is evaluated BmN cell viability, BmNPV is evaluated using qRT-PCR method in the proliferative ability handled in cell and uses Reed-
The appeal etc. of BmNPV in Muench Endpoint Dilution Method evaluation processing cell.The result shows that at through bmo-miR-3378 analog
After the BmN cell infection BmNPV of reason: cell viability is substantially reduced, and connect poison for 24 hours after difference reach the level of signifiance (p <
0.05)；The DNA copy number of BmNPV in cell is connecing significant decrease (p < 0.05) in malicious 72h；The titre of BmNPV is also significant
It reduces (p < 0.05).Correspondingly, after the BmN handled through bmo-miR-3378 mortifier carefully infects BmNPV, relevant index is equal
Inverse variation is presented.
The above results show bmo-miR-3378 can by regulate and control host cell apoptosis influence virus duplication with invade
Dye.Therefore, bmo-miR-3378 can be used as double regulation control molecule and be applied to promote or inhibit viral invading in host cell
Dye and proliferation.
Compared with prior art, the invention has the following advantages:
Present invention discover that bmo-miR-3378 expression quantity during silkworm BmN cell infection BmNPV significantly reduces, say
Bright its has specific function in virus and host's interaction；And it is experimentally confirmed bmo-miR-3378 and can pass through regulation
The apoptosis of BmN cell, so influence virus proliferation and infect, can be used as regulate and control baculovirus infection new tiny RNA,
Prevention and treatment antiviral to the silkworm in current sericultural production has weight with breeding and promotion silkworm biological reactor industrialized development
The realistic meaning wanted.
Detailed description of the invention
Fig. 1 is that BmNPV infects the qRT-PCR testing result influenced on bmo-miR-3378 expression quantity in Bombyx noriN cell
Schematic diagram.The result shows that, at virus infection early stage (in for 24 hours), bmo-miR-3378 expression quantity significant bombyx mori cell occurs in figure
It reduces (p < 0.05).
Fig. 2 is the qRT-PCR that bmo-miR-3378 analog influences bmo-miR-3378 expression quantity in Bombyx noriN cell
Testing result schematic diagram.The result shows that bombyx mori cell is after transfecting bmo-miR-3378 analog, bmo-miR-3378 expression quantity
Increase (p < 0.01) in extremely significant.This illustrates the effect that there is bmo-miR-3378 to be overexpressed for the processing of bmo-miR-3378 analog
Fruit.
Fig. 3 is the qRT-PCR that bmo-miR-3378 mortifier influences bmo-miR-3378 expression quantity in Bombyx noriN cell
Testing result schematic diagram.The result shows that bombyx mori cell is after transfecting bmo-miR-3378 mortifier, bmo-miR-3378 expression quantity
In extremely significant reduction (p < 0.01).This illustrates the effect that there is the processing of bmo-miR-3378 analog bmo-miR-3378 to inhibit silencing
Fruit.
Fig. 4 is bmo-miR-3378 analog on the Caspase Activity determination result of the apoptosis-induced influence of Bombyx noriN cell
Schematic diagram.The result shows that the level of apoptosis of bombyx mori cell is in virus infection morning after bmo-miR-3378 analog transfection processing
Phase (for 24 hours in) increases (p < 0.01) in extremely significant, after extend at any time and difference is no longer significant.This shows bmo-miR-3378 table
Up to amount increase can significant inducing cell to the apoptotic response of virus.
Fig. 5 is bmo-miR-3378 mortifier on the Caspase Activity determination result of the apoptosis-induced influence of Bombyx noriN cell
Schematic diagram.The result shows that the level of apoptosis of bombyx mori cell is after the virus infection after bmo-miR-3378 mortifier transfection processing
In significant decrease (p < 0.05) in 48h.This, which shows that bmo-miR-3378 expression quantity reduces, can significantly inhibit cell to virus
Apoptotic response.
Fig. 6 is MTT Activity determination result schematic diagram of the bmo-miR-3378 analog to Bombyx noriN cell effect of vigor.
The result shows that bombyx mori cell vigor is in significantly to increase after virus infection 12h after bmo-miR-3378 analog transfection processing
(p<0.05).This shows that bmo-miR-3378 expression quantity increases the vigor that can significantly increase cell.
Fig. 7 is MTT Activity determination result schematic diagram of the bmo-miR-3378 mortifier to Bombyx noriN cell effect of vigor.
The result shows that bombyx mori cell vigor is in virus infection for 24 hours afterwards in significant decrease after bmo-miR-3378 mortifier transfection processing
(p<0.05).This shows that bmo-miR-3378 expression quantity reduces the vigor that can be obviously reduced cell.
Fig. 8 is the qRT-PCR testing result schematic diagram that bmo-miR-3378 analog influences BmNPV proliferative ability.As a result
Show bombyx mori cell of the BmNPV infection through the transfection processing of bmo-miR-3378 analog, viral DNA copy number is after infection
It is in extremely significant reduction (p < 0.01) in 72h.This show bmo-miR-3378 expression quantity increase can significantly inhibit BmNPV DNA it is multiple
System is horizontal.
Fig. 9 is the qRT-PCR testing result schematic diagram that bmo-miR-3378 mortifier influences BmNPV proliferative ability.As a result
Show bombyx mori cell of the BmNPV infection through the transfection processing of bmo-miR-3378 mortifier, viral DNA copy number is after infection
Increase (p < 0.01) in extremely significant in 72h.This show bmo-miR-3378 expression quantity reduce can dramatically increase BmNPV DNA it is multiple
System is horizontal.
Figure 10 is the Reed-Muench testing result schematic diagram that bmo-miR-3378 analog influences BmNPV appeal.
The result shows that BmNPV infection, after the bombyx mori cell of bmo-miR-3378 analog transfection processing, viral titre levels are in pole
It significantly reduces (p < 0.01).This shows that bmo-miR-3378 expression quantity increases the appeal that can significantly affect BmNPV.
Figure 11 is the Reed-Muench testing result schematic diagram that bmo-miR-3378 mortifier influences BmNPV appeal.
The result shows that BmNPV infection, after the bombyx mori cell of bmo-miR-3378 mortifier transfection processing, viral titre levels are in pole
Significantly increase (p < 0.01).This shows that bmo-miR-3378 expression quantity reduces the appeal for being remarkably improved BmNPV.
Specific embodiment
The present invention is described further combined with specific embodiments below, but protection scope of the present invention is not limited only to
This.
The nucleotide sequence of bmo-miR-3378 is as shown in SEQ ID NO.1.It is thin used in case study on implementation of the present invention
Born of the same parents culture solution Sf-900TM II SFM (1 ×) and fetal calf serum (FBS) are purchased from U.S. Gibco company, and liposome is used in transfection
Lipofectamine LTX&PLUS Reagent and cell RNA, which are extracted, is purchased from U.S. Life with TRIzol Reagent etc.
Technologies company；Transcriptor First Strand cDNA Synthesis Kit, qRT- are employed in RNA reverse
PCR is extracted with FastStart Universal SYBR Green Master (ROX) and viral DNA and is used High Pure Viral
Nucleic Acid Kid etc. is purchased from Roche company, the U.S.；Apoptosis detection CaspACE Assay System
(Colrimetric) it is purchased from U.S. Promega company；Cell viability detection is purchased from green skies Bioisystech Co., Ltd with MTT.
Silkworm bmo-miR-3378 analog and its control, bmo-miR-3378 mortifier and its control are had by Shanghai Ji Ma pharmaceutical technology
Limit company designs and synthesizes；Particular sequence is as follows:
The detection method of following case study on implementation design is as follows:
1. the transfection of Bombyx noriN cell
(1) the good BmN cell about 1 × 10 of access state in 24 orifice plates5Behind a/hole, 27 DEG C of overnight incubations make cell
50%~70% confluent cultures hole；
(2) dilute bmo-miR-3378 analog: take 1.25 μ L, 20 μM of bmo-miR-3378 analog storing liquids in
In Nuclease-Free EP pipe, 50 μ L are diluted to Sf-900II SFM culture solution, gently pressure-vaccum 5 times mixings, incubation at room temperature
5min；
(3) it dilutes liposome: gently turning upside down and mix Lipofectamine LTX&PLUS reagent, rapid centrifugation to pipe
Bottom takes 2 μ L in Nuclease-Free EP pipe, is diluted to 50 μ L liposome dilutions with Sf-900II SFM culture solution, gently
Featheriness makes a call to 5 times and is allowed to mix, and is placed at room temperature for 5min；
(4) liposome and bmo-miR-3378 analog mixed liquor are prepared: by 50 μ L liposome dilutions and 50 μ L bmo-
MiR-3378 analog dilution mixes gently, and is incubated for 20min at room temperature；
(5) before the end of the incubation, the culture medium in orifice plate is absorbed, cleans cell culture with Sf-900II SFM culture solution
400 μ L culture is gently added dropwise based in cell culture well in hole；
(6) after incubation, liposome and bmo-miR-3378 analog mixed liquor are added drop-wise on cell；
Cell culture medium is changed into the Sf-900II SFM culture solution added with 10% fetal calf serum after (7) 27 DEG C of culture 5h；
The expression quantity of qRT-PCR method quantitative detection bmo-miR-3378 is used after (8) 27 DEG C of culture 48h.
In transfection experiment, 3 repeating holes are arranged in each transfection sample.Bmo-miR-3378 analog control sample-adding amount with
Bmo-miR-3378 analog is identical；Bmo-miR-3378 mortifier and its sample-adding amount of control are 2 times of analog.
The activation of 2.BmNPV-DsRED recombinant virus
It takes 3mL fresh and new T-25 Tissue Culture Flask is added added with the Sf-900II SFM culture solution of 10% fetal calf serum
In, then by after the good BmN cell piping and druming uniformly of growth conditions, 2mL is taken to be added drop-wise in T-25 Tissue Culture Flask；It is gently mixed
After cell suspension, it is placed at 27 DEG C and cultivates.When 80% confluent cultures bottle of cell, old culture solution is discarded, fresh medium is added,
And 50 μ L BmNPV-DsRED recombinant virus liquid are accessed, continue to cultivate after mixing gently.After cultivating 3~4d, to most cells
When swimming in culture solution, the culture solution for being rich in BmNPV particle is collected, removes the cell in virus liquid with 0.22 μm of membrane filtration
Fragment measures the titre of activation restrovirus with Reed-Muench Endpoint Dilution Method.
The qRT-PCR of 1 Bombyx noriN cell tiny RNA expression quantity of embodiment is detected
Take BmN cell inoculation in good condition in 24 porocyte culture plates, after 27 DEG C of 3~4h of culture, about 80% cell
It is paved with cell culture well, then accesses BmNPV-DsRED recombinant virus by MOI=0.15 amount, or transfect respectively according to the above method
Bmo-miR-3378 analog and its control, bmo-miR-3378 mortifier and its control, continue to cultivate after mixing.
1, cell total rna extracts
(1) culture solution in 24 well culture plates is exhausted, 500 μ L Trizol are added, place 10min after jiggling on ice,
Ensure that cell cracks completely with pipettor piping and druming, lysate is drawn in centrifuge tube；
(2) be stored at room temperature 5min, 0.1mL chloroform be added, acutely vibrates 15s, be placed at room temperature for 2min, 4 DEG C of 12000rpm from
Heart 15min draws supernatant into new RNase-free EP pipe；
(3) 0.25mL isopropanol is added, it is soft to mix, it is placed at room temperature for 10min, 4 DEG C of 12000rpm are centrifuged 10min, go
Clearly, it is seen that RNA precipitate；
(4) the 75% ethanol washing precipitating of 1.0mL DEPC water configuration is added, 4 DEG C of 7500rpm are centrifuged 5min, careful to exhaust
Liquid spontaneously dries, and the dissolution of 30 μ L DEPC water is added before being completely dried to RNA.The OD values such as 260nm and 280nm are measured, with inspection
RNA purity and concentration are tested, -80 DEG C of preservations are continued to employ.
2, cDNA is synthesized
Stem-loop primer (the sequence such as SEQ ID NO.2 of bmo-miR-3378 is separately designed, synthesized according to stem-loop method
It is shown), upstream primer (sequence is as shown in SEQ ID NO.3) and downstream primer (sequence is as shown in SEQ ID NO.4).Using family
Silkworm 5S rRNA (B5S) be reference gene, separately design and synthesize its upstream primer (sequence is as shown in SEQ ID NO.5) and under
It swims primer (sequence is as shown in SEQ ID NO.6).
It prepares and reverses by Roche company's T ranscriptor First Strand cDNA Synthesis Kit specification
Simultaneously response procedures are arranged in record reaction system liquid:
(1) new Nuclease-Free EP is taken to manage, sequentially add 1.0 μ g RNA, 1.0 μ L, 20 μM of Oligo dT or
10 μM of bmo-miR-3378Stem-loop primers of Random primer and 1.0 μ L, complement to 13 μ L with RNase-Free water, gently
Mixing is played in featheriness；
(2) 65 DEG C of water-baths are placed reaction liquid into and incubate 10min, are immediately placed on after the water bath is on ice；
(3) 4.0 μ L 5 × Transcriptor Reverse Transcriptase Reaction are sequentially added
Buffer, 0.5 μ L 40U/ μ L Protector RNase Inhibitor, 2.0 μ L dNTP (10mM each) and 0.5 μ L
20U/ μ L Transcriptor Reverse Transcriptase, gets rid of after mixing gently to tube bottom；
(4) reverse transcription reaction condition is as follows: 55 DEG C of 30min, 85 DEG C of 5min, and cDNA, -20 DEG C of guarantors are obtained after cooled on ice
It deposits.
3, qRT-PCR is detected
Using 50 × diluted cDNA as template, by Roche company Faststart Universal SYBR Green
Master (Rox) specification prepares the system of qPCR and response procedures is arranged:
New PCR pipe is taken to sequentially add FastStart Universal SYBR Green Master (ROX) 10 μ L, 10 μM
Each 1.5 μ L of 0.6 μ L and cDNA template of upstream and downstream primer, complements to 20 μ L with PCR-grade water.It is got rid of after mixing to tube bottom, is used
7300 fluorescence quantitative PCR instrument of ABI carries out qPCR detection.Reaction condition: 95 DEG C of initial denaturation 10min；95 DEG C of 15s, 60 DEG C of 60s, altogether
40 circulations.Simultaneously using B5S gene as reference gene, the experimental data for obtaining PCR amplification is analyzed using ABI 7300System,
Using 2-ΔΔCtExpression quantity digit difference is compared in the calculating of relative quantification method.
The result that stem-loop method qRT-PCR detects tiny RNA expression quantity is as shown in Figs. 1-3.Bombyx mori cell is in virus infection early stage
(in for 24 hours), bmo-miR-3378 expression quantity significantly reduces；And after transfecting bmo-miR-3378 analog or mortifier,
Significantly increasing or significantly reducing occur respectively in bmo-miR-3378 expression quantity.This shows that bmo-miR-3378 infects house in BmNPV
It is had a certain effect in silkworm cell processes.Meanwhile BmN cell bmo-miR-3378 overexpression/inhibition expression that the present invention constructs
Processing is effective with detection architecture.
2 bmo-miR-3378 of embodiment is on the Caspase Activity determination of the apoptosis-induced influence of Bombyx noriN cell
According to the above method to BmN cell transfecting bmo-miR-3378 analog or mortifier and its respectively after control 48h, with
MOI=0.15 amount accesses BmNPV-DsRED recombinant virus.
(1) connect poison culture 12h, for 24 hours, cell is collected after 48h and 72h respectively, 4 DEG C of 1000rpm are centrifuged 10min；
(2) cell precipitation is washed with ice cold PBS buffer, 4 DEG C of 1000rpm are centrifuged 10min；
(3) PBS buffer solution is discarded, Promega company CaspACE Assay System (Colrimetric) reagent is added
Cell is resuspended in cell pyrolysis liquid in box；
(4) being placed in about 2min at -80 DEG C freezes cell liquid completely, and then thaw about 20min on ice；
(5) after cell liquid thaws completely, 15min is stood on ice；
(6) freeze thawing 1 time is repeated to ensure that cell is cracked completely, and 4 DEG C of 13000rpm are centrifuged 20min, take supernatant for withering
Die detection or be stored in -80 DEG C it is spare.
After handling sample according to CaspACE Assay System (Colrimetric) kit specification, multi-functional
Absorbance value of each sample at 405nm is detected in microplate reader.
Testing result is as illustrated in figures 4-5.After bmo-miR-3378 analog transfection processing, the level of apoptosis of bombyx mori cell
Increase (p < 0.01) in extremely significant in virus infection early stage (for 24 hours in), after extend at any time and difference is no longer significant；Through bmo-
After miR-3378 mortifier transfection processing, the level of apoptosis of bombyx mori cell after the virus infection in 48h in significantly reduce (p <
0.05).This shows that the variation of bmo-miR-3378 expression quantity can significantly affect bombyx mori cell to the apoptotic response of virus.
MTT Activity determination of 3 bmo-miR-3378 of embodiment to Bombyx noriN cell effect of vigor
According to the above method to BmN cell transfecting bmo-miR-3378 analog or mortifier and its respectively after control 48h, with
MOI=0.15 amount accesses BmNPV-DsRED recombinant virus.
Connect poison culture 12h, for 24 hours, original fluid is discarded after 48h and 72h, the Sf- of 500 μ L fresh serum frees is added
25 μ L MTT are added in 900II SFM (1 ×) cell culture fluid, then every hole, in 27 DEG C of culture 4h after mixing；Add after sucking culture solution
Enter 750 μ L DMSO；Shaking table shakes 10min, detects the optical density in every hole at 490nm wavelength using multi-function microplate reader.
Testing result is as shown in fig. 6-7.After bmo-miR-3378 analog transfection processing, bombyx mori cell vigor is in virus
Infection is in significantly to increase afterwards for 24 hours；And after bmo-miR-3378 mortifier transfection processing, bombyx mori cell vigor is in virus infection
But in significant decrease after for 24 hours.After this shows that the variation of bmo-miR-3378 expression quantity can significantly affect bombyx mori cell virus infection
Vigor.
4 bmo-miR-3378 of embodiment detects the qRT-PCR that BmNPV proliferative ability influences
According to the above method to BmN cell transfecting bmo-miR-3378 analog or mortifier and its respectively after control 48h, with
MOI=0.15 amount accesses BmNPV-DsRED recombinant virus.
Poison culture 12h, for 24 hours, after 48h and 72h collect cell virus suspension respectively is being met, is taking 200 μ L by Roche company
The a small amount of purified virus genomic DNA of High Pure Viral Nucleic Acid Kit specification.
Design and synthesize the upstream primer (sequence is as shown in SEQ ID NO.7) and downstream primer (sequence for viral qPCR
Column are as shown in SEQ ID NO.8), using virus genom DNA as template, by above-mentioned Roche company Faststart
Universal SYBR Green Master (Rox) specification prepares qPCR system and response procedures is arranged, and carries out Viral Quantification
PCR detection.
The viral DNA sample concentration that measurement purifying obtains, calculates according to following equation and to contain in every microlitre of DNA sample
BmNPV-DsRED copy number (Copies/ μ L): DNA concentration (ng/ μ L) × 6.02 × 1023×10-9/(660×bases).Its
In, each base molecular weight is calculated by 660Da, and Avgadro constant is 6.02 × 1023, bases is DNA base number.With this
Calculating and obtaining the copy number of viral DNA standard product is 1.49 × 1011copies/μL.By viral DNA standard product ddH2O presses 10
After times gradient dilution, 1 μ L is taken to carry out qPCR reaction as template respectively, makes standard curve, and then to the viral DNA of each sample
Carry out quantitative analysis.
Testing result is as Figure 8-9.Bombyx mori cell of the BmNPV infection through the transfection processing of bmo-miR-3378 analog,
72h is interior in extremely significant reduction after infection for the DNA copy number of virus；And it infects through the transfection processing of bmo-miR-3378 mortifier
Bombyx mori cell, viral DNA copy number but increase in extremely significant in 72h after infection.This shows bmo-miR-3378 expression quantity
Variation can significantly affect proliferation of the BmNPV in bombyx mori cell.
5 bmo-miR-3378 of embodiment detects the Reed-Muench end dilution that BmNPV appeal influences
According to the above method to BmN cell transfecting bmo-miR-3378 analog or mortifier and its respectively after control 48h, with
MOI=0.15 amount accesses BmNPV-DsRED recombinant virus.Meeting poison culture 12h, for 24 hours, after 48h and 72h collect cytopathy respectively
Malicious suspension, it is continuous by 10 × work 10 times with cell culture fluid after the cell fragment in virus liquid is removed with 0.22 μm of membrane filtration
Gradient dilution.
100 μ l BmN cells (about 6 × 10 are all accessed in 96 orifice plates4A/hole), with containing 10% fetal calf serum at 27 DEG C
Sf-900II SFM culture solution culture 3h.Then, viral dilution is respectively connected in cell by column (i.e. 8 holes), every hole connects
100 μ l of poison；Add 100 μ l of culture solution in remaining 2 column cells, is set as compareing.It is placed at 27 DEG C and cultivates, daily under fluorescence microscope
It observes and records each column and the hole count of red fluorescence occurs, and calculate disease according to Reed-Muench method (Reed and Muench, 1938)
The TCID of poison50With MOI value.
Testing result is as shown in figs. 10-11.Bombyx mori cell of the BmNPV infection through the transfection processing of bmo-miR-3378 analog
Afterwards, viral titre levels are in extremely significant reduction；And infect after the bombyx mori cell of bmo-miR-3378 mortifier transfection processing,
The titre levels of virus are but in extremely significant increase.This shows that the variation of bmo-miR-3378 expression quantity can significantly affect BmNPV to family
The infecting potential of silkworm cell.
Claims (6)
1.bmo-miR-3378 promoting the application in the bombyx mori cell apoptosis by baculovirus infection, which is characterized in that described
Bombyx mori cell is Bombyx noriN cell；The baculoviral is nuclear polyhedrosis virus；The application is answering for non-treatment purpose
With.
Application of the 2.bmo-miR-3378 in the drug for preparing anti-silkworm baculovirus infection, which is characterized in that described rod-shaped
Virus is nuclear polyhedrosis virus.
3.bmo-miR-3378 analog is promoting the application in the bombyx mori cell apoptosis by baculovirus infection, and feature exists
In the bombyx mori cell is Bombyx noriN cell；The bmo-miR-3378 analog is double stranded RNA sequences, and nucleotide sequence is such as
Shown in SEQ ID NO.9 and SEQ ID NO.10；The baculoviral is nuclear polyhedrosis virus；The application is non-treatment mesh
Application.
Application of the 4.bmo-miR-3378 analog in the drug for preparing anti-silkworm baculovirus infection, which is characterized in that institute
Stating bmo-miR-3378 analog is double stranded RNA sequences, and nucleotide sequence is as shown in SEQ ID NO.9 and SEQ ID NO.10；
The baculoviral is nuclear polyhedrosis virus.
5.bmo-miR-3378 mortifier is inhibiting the application in the bombyx mori cell apoptosis by baculovirus infection, and feature exists
In the bombyx mori cell is Bombyx noriN cell；The nucleotide sequence of the bmo-miR-3378 mortifier such as SEQ ID NO.13
It is shown；The baculoviral is nuclear polyhedrosis virus.
The application that 6.bmo-miR-3378 mortifier promotes recombinant baculovirus proliferation and infected in silkworm biological reactor,
It is characterized in that, the nucleotide sequence of the bmo-miR-3378 mortifier is as shown in SEQ ID NO.13；The baculoviral is
Nuclear polyhedrosis virus.
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