CN105535975B - Application of the vesicle-associated membrane albumen 1 in prevention enterovirns type 71 infection - Google Patents
Application of the vesicle-associated membrane albumen 1 in prevention enterovirns type 71 infection Download PDFInfo
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Abstract
The present invention relates to field of biomedicine technology, are novel targets and application that a kind of anti-enterovirns type 71 infects.The present invention is using human neuroblastoma cells (SH SY5Y) as target cell, the expression of target cell host protein is lowered using RNA perturbation techniques, to find the host factor that can effectively inhibit EV71 infection human neuroblastoma cells (SH SY5Y), the damage of pre- preventing virus infection central nervous system and reduction to nervous system.The present invention is found through experiments that vesicle-associated membrane albumen 1 (vesicle associated membrane protein 1, VAMP1) plays an important role in EV71 infection SH SY5Y, lowers the expression of VAMP1, can substantially inhibit the infection of EV71.The present invention provides applications of the VAMP1 in prevention or treatment enterovirns type 71 infection medicine is prepared.
Description
Technical field
The present invention relates to field of biomedicine technology, are novel targets and application that a kind of anti-enterovirns type 71 infects.
Background technology
Enterovirns type 71 (enterovirus 71, EV71) belongs to Picornaviridae enterovirus genus people's enteron aisle
A viroids are one of main pathogens for causing hand-foot-and-mouth disease (Hand-foot-mouth disease, HFMD).At present, hand
Sufficient stomatosis is broken out in the multiple areas in the world and prevalence, the especially Asian-Pacific area.In China, brothers are broken out from 2008 Nian Ji great provinces and cities
Since stomatosis epidemic situation, the sick number of the infected and the death rate remain high always, and number of falling ill per annual report is more than 1,000,000, extremely
Die several nearly 1000.Hand-foot-and-mouth disease principal pathogenetic crowd be 5 years old Infants Below, clinical manifestation for fever, hand, foot, buttocks with
And there are the symptoms such as bleb in the positions such as oral mucosa；A small number of infants can develop into patient with severe symptoms, central nervous system occur
(central nervous system, CNS) lesion, including aseptic meningitis, brainstem encephalitis, encephalomyelitis and neural source
Property pulmonary edema etc., serious threat infant life and health [Solomon T1, Lewthwaite P, Perera D,
Cardosa MJ,McMinn P,Ooi MH.Virology,epidemiology,pathogenesis,and control of
enterovirus 71.Lancet Infect Dis.2010Nov；10(11):778-90.].Hand-foot-and-mouth disease particularly severe disease
Example is mostly caused by enterovirns type 71 (EV71) infects.However, on EV71 the treatment of hand-foot-and-mouth disease is caused to there is no specifically at present
Efficient antiviral drugs clinically still based on symptomatic treatment, also comes out in terms of prevention without effective vaccine.
EV71 infection has Neural invasion, easily causes serious CNS diseases and its complication, this is also to cause hand-foot-and-mouth disease
Patient evolution is severe even main causes of death.Wherein, brain stem is position [Tee, K.K., the et most easily infected by EV71
al.Evolutionary genetics of human enterovirus 71:origin,population dynamics,
natural selection,and seasonal periodicity of the VP1gene.J Virol,2010.84(7):
P.3339-50.], research is confirmed to be able to detect that EV71 genes at positions such as the brain stem neurons of EV71 patients with severe symptoms and resisted
Former presence, it was demonstrated that EV71 can enter CNS.EV71 destroys blood-brain barrier or through the reverse aixs cylinder of peripheral nerve approach through blood dissemination
Conveying infection CNS.But [Solomon T, Lewthwaite is still not clear in the mechanism of central nervous system symptom caused by EV71
P,Perera D,Cardosa MJ,Mcminn P,Ooi MH.Virology,epidemiology,pathogenesis,and
control of enterovirus 71.Lancet Infect Dis.2010；10:778-90.].Neuroblastoma cell
(SH-SY5Y) it is nervous tissue source, there is certain nerve cell characteristic.Therefore, the mechanism of EV71 infection SH-SY5Y is verified
And new antiviral target spot is found with this, it is most important for the central nervous system infection caused by prevention EV71.
In order to which effectively infection cell, virus must utilize the membrane molecule of host cell and its vesicular transport system completion pair
The invasion of host cell could be replicated and release the progeny virion of infectious in the cell.Therefore, as disease
The primary link of poison infection host cell, cell entry have become the important target of antiviral drugs screening.Research shows EV71
Different types of target cell, such as EV71 infection Jurkat T lymphs can be invaded using different host factor and route of infection
Endocytic pathway (caveolar-dependent endocytosis) [Lin HY, Yang that cell line is mainly relied on using alveole
YT,Yu SL,et al.Caveolar endocytosis is required for human PSGL-1-mediated
enterovirus 71infection.J Virol.2013,87(16):9064-76.].At present, SH- is infected on EV71
The approach and mechanism of SY5Y is still unclear.
The key molecule that Virus entry target cell is mainly transported by means of host cell Self substances are participated in, these hosts are thin
After birth transport molecule is played an important role in the formation of vesica, endocytosis and secretion in the cross-film matter transportation of cell.Such as
Metalloproteinases has adjusted cell adherence, the degradation of membrane molecule；Caveolin family molecule caveolin-1 (CAV1),
Caveolin-2 (CAV2), caveolin-3 (CAV3) are by matter transportation to golgiosome；Flotillin molecules can be with Lipid Rafts
It is swallowed in interior protein molecular interaction simultaneously intracellular；Vesicle-associated membrane albumen 1 participate in synapses synaptic vesicle exocytosis with
Reuptake process；Gtpase activating protein molecule GRAF1 has important adjusting in the vesicle transport of clathrin independent form
Act on (Jason Mercer, Mario Schelhaas, et al.Virus Entry by Endocytosis.Annu Rev
Biochem.2010；79:803-833.).In these molecules, caveolin-1 is proved to take part in EV71 infection Jurkat T
The process of lymphocyte, and effect of other molecules in EV71 infection does not have been reported that also.
Vesicle-associated membrane albumen 1 (vesicle-associated membrane protein 1, VAMP1) is cynapse capsule
Memebrane protein on bubble, VAMP1 are primarily present in presynaptic membrane, played an important role in cynapse transmission (Yun Liu,
Yoshie Sugiura andWeichun Lin.The role of Synaptobrevin1/VAMP1in Ca2+-
triggered neurotransmitter release at the mouse neuromuscular junction.The
Journal of Physiology.2011；pp:1603–1618.).Recent study it has also been found that VAMP1 molecules virus sense
It plays an important role during dye, Jing Zhang et al. have found VAMP1 molecular regulations infection with hepatitis C virus
Huh-7 cells (Jing Zhang, Osamu Yamada, Takashi Sakamoto, Hiroshi Yoshida, Takahiro
Iwai,Yoshihisa Matsushita,Hideo Shimamura,Hiromasa Araki and Kunitada
Shimotohno.Down-regulation of viral replication by adenoviral-mediated
expression of siRNA against cellular cofactors for hepatitis C
virus.Virology.320(2004):135–143.).Human rhabdomyosarcoma's RD cells are the sensitive cell lines of EV71, however
Khairunnisa etc. is screened by genomic library finds that VAMP1 molecules do not influence infection of the EV71 to RD cells
(Hussain KM1,Leong KL,Ng MM,Chu JJ.The essential role of clathrin-mediated
endocytosis in the infectious entry of human enterovirus 71.J Biol Chem.2011；
286(1):309-21.)。
There is presently no it is any on VAMP1 molecules in EV71 infects SH-SY5Y the report that acts on, for the molecule into
Row further investigation can not only promote the understanding to EV71 infection and pathogenic mechanism, or prevention and treatment EV71 infection carries
For new thinking and target spot.
The content of the invention
It is an object of the invention to provide a kind of novel targets of anti-enterovirns type 71 infection.
Another object of the present invention is to provide vesicle-associated membrane albumen 1 (VAMP1) new application of molecule, particularly anti-
Application in enterovirns type 71 infection.
The third object of the present invention is the siRNA for providing interference VAMP1 developed by molecule.
The present invention main technical schemes be：
Using human neuroblastoma cells (SH-SY5Y) as target cell, target is lowered using RNA perturbation techniques by the present invention
The expression of cell host albumen, to find the host that can effectively inhibit EV71 infection human neuroblastoma cells (SH-SY5Y)
The factor, so as to reduce nervous system infection with destroying.This experimental selection one group of host cell transmembrane transporter molecules are sieved
Choosing, these molecules are played an important role, they are often in the endocytosis of vesica and secretion in the cross-film matter transportation of host cell
And easily by viral " abduction " and the molecule that utilizes in virus infection.These molecules include：Metalloproteinases 10
(ADAM10), caveolin-1 (CAV1), caveolin-2 (CAV2), caveolin-3 (CAV3), Flotillin albumen 1
(FLOT1), Flotillin albumen 2 (FLOT2), gtpase activating protein (GRAF1), synaptotagmin 1 (SYT1), synaptic knob
Hop protein 2 (SYT2).Obtain complete sequence and mRNA sequence by retrieving NCBI GeneBank, using existing Internet resources and
Popular software carries out biological analysis to these genes, then the target sequence that code area is selected to be designed as siRNA designs
SiRNA, by lowering these molecules, to observe the influence to EV71 infection.
We have found that vesicle-associated membrane albumen 1 (vesicle-associated membrane protein 1, VAMP1)
It plays an important role in EV71 infects SH-SY5Y, lowers the expression of VAMP1, can substantially inhibit the infection of EV71.
The first aspect of the present invention provides vesicle-associated membrane albumen 1 (VAMP1) and is used as a kind of anti-enterovirns type 71 sense
The novel targets of dye.
The vesicle-associated membrane albumen 1 (VAMP1), GENBANK ID：NM_014231.
The second aspect of the present invention provides vesicle-associated membrane albumen 1 (VAMP1) and is preparing prevention or treatment enterovirus
Application in 71 type infection medicines.
Further, the present invention also provides vesicle-associated membrane albumen 1 (VAMP1) to prepare prevention or treatment hand-foot-and-mouth disease medicine
Application in object.
Vesicle-associated membrane albumen 1 (VAMP1) of the present invention is preparing prevention or treatment enterovirns type 71 infection medicine
Application in object, the drug specifically refer to inhibit or lower the reagent of the expression quantity of VAMP1.
The reagent for the expression quantity that VAMP1 is lowered in the inhibition can be siRNA, shRNA, comprising siRNA, shRNA
Recombinant vector (such as plasmid).
The third aspect of the present invention, the RNA interfering the present invention provides vesicle-associated membrane albumen 1 (VAMP1) are being prepared in advance
Anti- or application in treatment enterovirns type 71 infection medicine or vesicle-associated membrane albumen 1 (VAMP1) are preparing prevention or treatment
Application in hand-foot-and-mouth disease drug, the drug are RNA interfering (siRNA), and sequence is as follows：
CAUGACCAGUAACAGACGAUU(SEQ ID NO:1)
CAUCGUGGUAGUUAUUGUAUU(SEQ ID NO:2)
GCGUUCAUUUGCACUCUGUUU(SEQ ID NO:3)
Wherein, with such as SEQ ID NO:SiRNA shown in 1 lowers the expression quantity best results of VAMP1, and reduces EV71 pairs
The infection of SH-SY5Y cells is the most apparent.
The present invention screens the new host cellular molecules VAMP1 that can inhibit EV71 infection SH-SY5Y cells.
After VAMP1 gene deregulations, the normal physiological function of cell is not influenced, but substantially inhibits senses of the EV71 to SH-SY5Y cells
Dye.
Therefore nervous system damage of the present invention for clinical prevention and treatment caused by EV71 infects provides new target
Point and therapeutic scheme.
Description of the drawings
Fig. 1 detects to transfect the jamming effectiveness after effective siRNA and cytotoxicity, and primary axis represents that interference is imitated in figure
Rate, secondary axis represent the influence to cytotoxicity；
CTRL：The SH-SY5Y groups of cells (ghost group) of any siRNA is not transfected；
NT：Transfect the SH-SY5Y groups of cells (negative control group) of non-targeting siRNA；
siRNA：Transfection is for the SH-SY5Y groups of cells (experimental group) of the siRNA of each target gene.
Fig. 2 is the influence to EV71 infection after each host molecule of immuno-fluorescence assay is lowered, and wherein A is each molecule of downward
Afterwards to the fluoroscopic examination figure of viral infection, B is to lower the inhibiting rate figure infected after each molecule virus；
CTRL：The SH-SY5Y groups of cells (ghost group) of any siRNA is not transfected；
NT：Transfect the SH-SY5Y groups of cells (negative control group) of non-targeting siRNA；
siRNA：Transfection is for the SH-SY5Y groups of cells (experimental group) of the siRNA of each target gene.
Fig. 3 is the influence to EV71 infection after VAMP1 is lowered, and wherein A is the table that Western Blot detect VAMP1 albumen
Up to figure, B is the cytopathic effect figure of observation EV71, and C is detection EV71 virus spirograms；
CTRL：The SH-SY5Y groups of cells (ghost group) of any siRNA is not transfected；
NT-CTRL：Transfect the SH-SY5Y groups of cells (negative control group) of non-targeting siRNA；
VAMP1：Transfection is for siRNA (the SEQ ID NO of VAMP1 genes:1) SH-SY5Y groups of cells.
Fig. 4 influences to scheme for the jamming effectiveness after the disturbance sequence of transfection VAMP1 molecules and on EV71 is infective, A
Figure is detected for the mRNA level in-site of VAMP1 genes, B is EV71 virus quantities detection figure；
CTRL：The SH-SY5Y groups of cells (ghost group) of any siRNA is not transfected；
NT-CTRL：Transfect the SH-SY5Y groups of cells (negative control group) of non-targeting siRNA；
VAMP1-1：Transfection is for siRNA (the SEQ ID NO of VAMP1 genes:1) SH-SY5Y groups of cells；
VAMP1-2：Transfection is for siRNA (the SEQ ID NO of VAMP1 genes:2) SH-SY5Y groups of cells；
VAMP1-3：Transfection is for siRNA (the SEQ ID NO of VAMP1 genes:3) SH-SY5Y groups of cells.
Specific embodiment
In conjunction with embodiment and attached drawing, the present invention is described in detail, but the implementation of the present invention is not limited only to this.
The reagents and materials used in the present invention are commercially available or can be prepared by literature method.Tool is not specified in the following example
The experimental method of concrete conditions in the establishment of a specific crime, usually according to normal condition such as Sambrook et al.《Molecular cloning：Lab guide》(New
York：Cold Spring Harbor Laboratory Press, 1989) condition described in or according to normal condition or
According to the condition proposed by manufacturer.Unless otherwise stated, otherwise percentage and number are calculated by weight.
Embodiment 1：
1 design, the specific siRNA sequence for synthesizing each host cellular molecules.
1.1 are directed to each target gene, and retrieval NCBI GeneBank obtain complete sequence and mRNA sequence, and utilization is existing
Internet resources and popular software carry out biological analysis, the target sequence that code area is selected to be designed as siRNA to each target gene.
With reference to siRNA design principles, and the blast functions of passing through GeneBank databases are compared with human genomic sequence, really
It protects without homology；Exclude 5 ' continuous 8 bases in end of aitisense chains and the potential siRNA of other genes pairing；It excludes any
One section of continuous 14 base and the potential siRNA of other genes pairing.And Pre-Evaluation measure is carried out using design software, select 3
A optimal kinetic parameter target spot enters subsequent experimental flow, and each gene synthesizes 3 interference sequences, is shown in Table 1 altogether.
The synthesis of 1.2 single-stranded siRNA is completed with purifying by Invitrogen companies.
The design of table 1.siRNA target spots
2 siRNA sequences are screened to be identified with interference effect
2.1 RNA transfection
Transfection procedure is with reference to 2000 specifications of Lipofectamine
1) shift to an earlier date 12-16 it is small when by SH-SY5Y cells (purchased from ATCC, preserving number：ATCC CRL-2266) to be layered on 24 holes thin
It is cultivated on born of the same parents' culture plate so that cell density is 80%-90% during transfection.
2) take 2 μ L Lipofectamine 2000 add in 50 μ L opti-MEM in and soft mixing, be incubated at room temperature 5 points
Clock；It is another to take the RNA interfering and 50 μ L opti-MEM mixing that 5 μ L concentration are 5 μM.It, will be diluted after incubation
2000 transfection reagents of Lipofectamine are added in diluted RNA, and soft pressure-vaccum mixing.After being incubated at room temperature 20min, add in
In SH-SY5Y cells, 400 μ L opti-MEM are added so that the final concentration of 50nM of RNA.
3) replaced when 6-8 is small after transfecting and contain dual anti-fresh culture.
2.2 real-time fluorescence quantitative PCRs (RT-PCR) detect the mRNA level in-site of each host molecule
1) TRIzol extracts the total serum IgE of control group and interference group cell, is as follows：
Transfect 48 it is small when after, go culture supernatant, in cell add in 1ml TRIzol, be sufficiently mixed lysis at room temperature cell 3-
5 minutes.The chloroform of 1/5 volume is added in, is vigorously mixed manually 15 seconds.The heart is left in 4 DEG C, 12,000 15 minutes.Take upper strata aqueous phase
And be transferred in new EP pipes, isometric isopropanol is added in, is sufficiently mixed, precipitation at room temperature 10 minutes.It is left in 4 DEG C, 12,000
The heart 10 minutes.Supernatant is abandoned, adds in 75% ethyl alcohol of 1ml precoolings.It is centrifuged 5 minutes in 4 DEG C, 12,000.Supernatant is fully abandoned, room temperature is dried in the air
Dry RNA precipitate adds in DEPC processing water dissolution precipitations, obtains total serum IgE.
2) cDNA of control group and interference group cell is obtained using takara reverse transcription reagent box, is as follows：
Following reaction system is added in PCR pipe,
Soft mixing mixing, is placed in 37 DEG C and reacts 15 minutes, is subsequently placed in 85 DEG C of heating, 5 seconds inactivation reverse transcriptases.
3) fluorescence quantitative RT-RCR detects
It is reacted using the SYBR Premix Ex Taq kits of takara, reaction system is as follows,
Two-step method amplification is carried out using Rotor Gene 3000A instruments, 95 DEG C of pre-degeneration 2min carry out 40 PCR and follow
Ring, 95 DEG C 5 seconds, 60 DEG C 30 seconds.
3 cytotoxicity experiments
The influence of cell proliferation after transfection siRNA is detected using CCK-8 methods, is as follows：
Exponential phase cell is collected, 96 orifice plates are inoculated in the density in every 3000, hole.After cell pellet overnight is adherent, turn
Contaminate each siRNA, when culture 48 is small after detect cell proliferative conditions.Original culture medium is discarded, is added in per hole containing 10 μ L CCK-8
110 μ L of fresh culture use multi-function microplate reader in each hole absorbance of 450nm wavelength detectings after cultivating 3h.The independent weight of experiment
It is 3 times multiple, calculate average value.
4 EV71 virus infection SH-SY5Y cells
The EV71 virus infection experiments of 4.1 SH-SY5Y cells
After SH-SY5Y cell transfectings RNA 72 it is small when, carry out EV71 virus infection experiments.Culture supernatant is suctioned out, uses pre-temperature
PBS rinses 2 times are inoculated with EV71 with the virus quantity of MOI=0.1, and 37 DEG C are incubated after 2h and discard virus liquid, and with pre-temperature PBS rinses 3
It is secondary, it adds in fresh culture and continues to cultivate.
4.2 immunofluorescence dyeings detect EV71 antigen presentations
Continue to cultivate 48h after SH-SY5Y cell infection viruses, using the expression of immuno-fluorescence assay viral antigen, tool
Body step is as follows：
1) cell is fixed：Culture solution in 96 orifice plates is removed, adds in PBS cleaning cell 2 times, it is pre- to add in 100 μ l per hole
Cold methanol fixes 20min under the conditions of -20 DEG C, with the PBS cleaning cell 3 times of precooling.
2) permeable membrane：Cell per well after fixation adds in 100 μ l 0.1%TritonX-100, is incubated at room temperature 15min, with pre-
Cold PBS is washed 3 times.
3) close：100 μ l 3%BSA are added in per hole, are incubated 1h at room temperature.
4) primary antibody is incubated：EV71 specific murines source monoclonal antibody 10F0 (1 is added in per hole:2000 dilutions) 100 μ l, incubation at room temperature
1h is washed 3 times with the PBS of precooling.
5) secondary antibody is incubated：The anti-mouse IgG (1 of 488 fluorescent markers of AF is added in per hole:1000 dilutions) 100 μ l, room temperature, which is protected from light, incubates
1h is educated, washing 2 times is protected from light with the PBS of precooling.
6) nucleus is marked：Nucleus fluorescent dye DAPI (1 is added in per hole:5000, PBS dilutions), room temperature is protected from light incubation
15min is protected from light washing 3 times with the PBS of precooling.
7) detected under fluorescence microscope and calculate green 488 positive cell clone numbers of AF.
4.3 protein immunoblot.
(1) total protein of control group and VAMP1 interference group SH-SY5Y cells is extracted respectively with protein lysate.
(2) 30ug albumen is added to electrophoresis in the polyacrylamide gel of 12.5% concentration respectively after quantification of protein, and
Interception respective strap is gone to electroporation on pvdf membrane.
(3) non-specific sites of albumen are closed with 5% skim milk, are then closed with VAMP1 antibody, and 4 DEG C are overnight,
It is washed three times with TBST buffer solutions, washes away primary antibody.
(4) and then with HRP when the secondary antibody incubation at room temperature 2 marked is small, then washed three times with TBST buffer solutions.
(5) finally, developing solution colour developing and photographic analysis are utilized
EV71 virus quantities in 4.4RT-PCR detection cells
Continue to cultivate 48h after SH-SY5Y cell infection viruses, the total of control group and interference group cell is extracted using TRIzol
RNA, and reverse transcription obtains cDNA, and EV71 virus quantities are detected by RT-PCR.Specific steps are the same as shown in 2.2.
Experimental result：
1 design synthesizes and screens effective siRNA
For each objective gene sequence, we devise multiple RNA interfered target sequences, and are carried out using design software
Pre-Evaluation measures, and 3 optimal kinetic parameter target spots is selected to enter subsequent experimental flow, each gene synthesizes 3 interference altogether
Sequence, as shown in table 1.
Using the method for in-vitro transfection, the RNA interfering of each gene is transfected into SH-SY5Y cells, is passed through after 48h
RT-PCR methods detect the jamming effectiveness of each RNA interfering, finally screen the optimal siRNA sequence of interference effect (overstriking sequence in table 2
Row) subsequent experimental is carried out, jamming effectiveness is as shown in table 2.
2 RT-PCR methods of table detect downward efficiency of the siRNA interference sequences to related host gene
Note：CTRL：The SH-SY5Y groups of cells (ghost group) of any siRNA is not transfected
NT：Transfect the SH-SY5Y groups of cells (negative control group) of non-targeting siRNA
siRNA：Transfection is for the SH-SY5Y groups of cells (experimental group) of the siRNA of each target gene.
Jamming effectiveness and cytotoxicity detection after 2 siRNA interference
The effective siRNA for each host molecule picked out transfects SH-SY5Y cells, and 48h passes through RT-PCR after transfection
Method detects the jamming effectiveness of each RNA interfering, while using the influence to SH-SY5Y cytotoxicities after CCK8 detection transfections.
The results are shown in Figure 1, transfects effective siRNA groups compared with CTRL groups, can substantially inhibit phase after transfecting each siRNA
Answer the expression (P ＜ 0.01) of gene.Transfect VAMP1siRNA (SEQ ID NO:1) inhibition efficiency can reach 77%.
Cytotoxicity experiment does not generate apparent cytotoxicity (P ＞ 0.05) after showing each siRNA transfections, to thin
The normal physiological function of born of the same parents does not have an impact, available for subsequent experimental.
To the influence of EV71 virus infection after 3 siRNA interference
Effective siRNA of each host molecule is transfected after lowering the expression of host cell correlation molecule, to infect same dose
EV71 viruses, after infecting 48h, to the influence of EV71 infection after being lowered using each host molecule of immuno-fluorescence assay, find
Compared with the control group, VAMP1siRNA (SEQ ID NO are transfected:1) after making VAMP1 gene deregulations, hence it is evident that reduce EV71 to SH-
The infection (Fig. 2A) of SY5Y cells.It is found by calculating virus quantity, the inhibiting rate of virus is reached after VAMP1 gene deregulations
79.25%, and there is no the apparent infection (P ＞ 0.05) (Fig. 2 B) for inhibiting EV71 to SH-SY5Y cells for the downward of remaining molecule.
For the inhibitory action that clear and definite VAMP1 infects EV71, after VAMP1 molecules siRNA is transfected, pass through Western blot
The expression of VAMP1 protein moleculars is detected, and observes cytopathy situation after EV71 is infected and EV71 is detected by RT-PCR
Virus quantity.The results show that transfection VAMP1 molecules siRNA (SEQ ID NO:1) after, VAMP1 protein moleculars can substantially be inhibited
It expresses (Fig. 3 A).Compared with the control group, after VAMP1 protein expressions are lowered, can inhibit in cytopathy and SH-SY5Y cells
Virus quantity is also remarkably decreased (Fig. 3 B, C), consistent with immuno-fluorescence assay result.These results indicate that with control cell phase
Than EV71 is decreased obviously the infection ability of SH-SY5Y cells after lowering VAMP1 genes, and virus quantity is reduced.
Further, influence of the siRNA observations of three VAMP1 molecules to viral infection is transfected respectively.The result shows that not
Same siRNA is different (Fig. 4 A) to the downward efficiency of VAMP1 molecules, wherein siRNA (SEQ ID NO:1) jamming effectiveness is most
Height, it is consistent with the result of front.It is infective on EV71 after detection interference to influence to find, siRNA pairs of three VAMP1 molecules
The inhibiting rate of virus infection can reach more than 64%, and increasing with the downward efficiency to VAMP1 molecules, and EV71 is felt
The inhibiting rate of dye also in rise (Fig. 4 B), prompts VAMP1 molecules to be played an important role in EV71 infects SH-SY5Y.Therefore,
VAMP1 can be as inhibition EV71 to new host's target spot of SH-SY5Y cell infections.
Pass through more than the results show：The present invention screens that can to inhibit EV71 infection one of SH-SY5Y cells new
Host cellular molecules VAMP1.After VAMP1 gene deregulations, the normal physiological function of cell is not influenced, but is substantially inhibited
Infection of the EV71 to SH-SY5Y cells.Therefore nervous system of the present invention for clinical prevention and treatment caused by EV71 infects
Damage provides new target spot and therapeutic scheme.
The preferred embodiment of the invention is illustrated above, but the invention be not limited to it is described
Embodiment, those skilled in the art can also make a variety of equivalent on the premise of without prejudice to the invention spirit
Modification or replacement, these equivalent modifications or replacement are all contained in the application claim limited range.
Claims (6)
1. can inhibit or lower vesicle-associated membrane albumen 1 expression quantity reagent prepare prevention or treatment enterovirns type 71
Application in infection medicine.
2. can inhibit or lower vesicle-associated membrane albumen 1 expression quantity reagent prepare prevention or treatment hand-foot-and-mouth disease drug
In application, caused by the hand-foot-and-mouth disease is enterovirns type 71.
3. application according to claim 1, which is characterized in that described to inhibit or lower vesicle-associated membrane albumen 1
The reagent of expression quantity be siRNA, shRNA of vesicle-associated membrane albumen 1 or the recombinant vector comprising siRNA, shRNA.
4. application according to claim 1, which is characterized in that described to inhibit or lower vesicle-associated membrane albumen 1
Expression quantity reagent be vesicle-associated membrane albumen 1 RNA interfering, the sequence of the RNA interfering is selected from following any：
CAUGACCAGUAACAGACGAUU(SEQ ID NO:1)、
CAUCGUGGUAGUUAUUGUAUU(SEQ ID NO:2)、
GCGUUCAUUUGCACUCUGUUU(SEQ ID NO:3)。
5. application according to claim 2, which is characterized in that described to inhibit or lower vesicle-associated membrane albumen 1
The reagent of expression quantity be siRNA, shRNA of vesicle-associated membrane albumen 1 or the recombinant vector comprising siRNA, shRNA.
6. application according to claim 2, which is characterized in that described to inhibit or lower vesicle-associated membrane albumen 1
Expression quantity reagent be vesicle-associated membrane albumen 1 RNA interfering, the sequence of the RNA interfering is selected from following any：
CAUGACCAGUAACAGACGAUU(SEQ ID NO:1)、
CAUCGUGGUAGUUAUUGUAUU(SEQ ID NO:2)、
GCGUUCAUUUGCACUCUGUUU(SEQ ID NO:3)。
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| CN112057622B (en) * | 2019-06-11 | 2021-07-06 | 中国人民解放军第二军医大学 | Application of ATP (adenosine triphosphate) binding cassette transmembrane transport subfamily C member 3 in preparation of medicine for preventing and treating enterovirus 71infection |
| CN112057621B (en) * | 2019-06-11 | 2021-07-02 | 中国人民解放军第二军医大学 | Application of solute carrier family 7member 7in preparation of medicine for preventing and treating enterovirus 71infection |
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| WO2008036060A2 (en) * | 2005-04-05 | 2008-03-27 | Allergan, Inc. | Clostridial toxin activity assays |
| CN103893748A (en) * | 2012-12-27 | 2014-07-02 | 上海吉美生物工程有限公司 | Novel vaccine for hand-foot-and-mouth disease and a preparation method thereof |
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| WO2008036060A2 (en) * | 2005-04-05 | 2008-03-27 | Allergan, Inc. | Clostridial toxin activity assays |
| CN103893748A (en) * | 2012-12-27 | 2014-07-02 | 上海吉美生物工程有限公司 | Novel vaccine for hand-foot-and-mouth disease and a preparation method thereof |
Non-Patent Citations (2)
| Title |
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| Enterovirus 71 induces apoptosis of SH‑SY5Y human neuroblastoma cells through stimulation of endogenous microRNA let‑7b expression;XILING DU et al;《MOLECULAR MEDICINE REPORTS》;20150313;第12卷;第953-959页 * |
| 肠道病毒71型复制周期研究进展;陈生林等;《中国病毒病杂志》;20150331;第5卷(第2期);第144-149页 * |
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