CN108841822A - Nanometer selenium supported V P1 gene siRNA and the preparation method and application thereof - Google Patents
Nanometer selenium supported V P1 gene siRNA and the preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a kind of nanometer selenium supported V P1 gene siRNA, the partial size of the nanometer selenium supported V P1 gene siRNA is smaller, can avoid immune system and reach the smallest capillary, extend the residence time in blood flow;And its target with higher selectivity, longer half-life period.The gene loci of the siRNA interference is the 157th nucleotide;The siRNA sequence is:Sense 5 '-ggcaucaucaaaugcuagutt-3 ', Antisense 5 '-acuagcauuugaugaugcctt-3 '.The present invention also provides the preparation method and application of above-mentioned nanometer selenium supported V P1 gene siRNA.
Description
Technical field
The present invention relates to nanometer selenium supported V P1 gene siRNA, especially nanometer selenium supported V P1 gene siRNA and its preparations
Method and application.
Background technique
Hand-foot-and-mouth disease is the common transmittable disease being caused by enterovirus, fallen ill with infant based on, with fever and hand, foot,
The fash or bleb at the positions such as oral cavity are main feature.Most patients symptom is slight, but small number of patients can complicated myocardial it is scorching,
The mortality complication such as pulmonary edema, AFP Cases, aseptic meningitis, encephalitis, individual children with serious disease disease progressions are fast
Speed easily causes death.Hand-foot-and-mouth disease since first Chinese large-scale outbreak, often there is different degrees of stream from 2008 every year
Row, often results in various regions kindergarten and is forced the garden of suspending classes, and carrys out huge negative effect to society and economy-zone.Enterovirns type 71
It (EV71) is the main pathogen for causing hand-foot-and-mouth disease.Under the morbidity and mortality of hand-foot-and-mouth disease have always been high not nearly ten years
Principal element is that there are many genotype for EV71 virus, and constantly variation, the gene mutation in certain sites can cause viral cause in the groove
The change of characteristic of disease makes the prevention and treatment of hand-foot-and-mouth disease be faced with huge pressure.It is clinical main there is presently no effective drug therapy
It is that the ill and supportive treatment, therefore, the vaccine and effective drug for preventing and treating hand-foot-and-mouth disease are a problem to be solved.
EV71 viral inactivation vaccine is in the clinical official written reply of acquirement in 2015, but the disease incidence of EV71 virus is not in recent years
Decline, main cause have:Vaccine strain is using C4 hypotype EV71 Strain, and there may be to make the characteristics of variation for clinical epidemic strain
It is bad at the preventive effect of vaccine;In addition, 6th month neutralize antibody titers are begun to decline after vaccine immunity, the protection period is limited.
There is no active drug for hand-foot-and-mouth disease at present, clinically still with suit the medicine to the illness and supportive treatment based on, therefore, effective anti-hand-foot-and-mouth-disease
The drug research of cause of disease, the drug research of especially wide spectrum anti-hand-foot-and-mouth-disease cause of disease have important meaning.
RNA perturbation technique can play a role for viral genome conservative region, to limit to a certain extent
Virus generates the ability for escaping mutant strain.Possessed by RNA perturbation technique to the specificity of target gene silencing effect, high efficiency,
Stability and not changing the characteristics such as host genome, the application for being RNA perturbation technique in antiviral therapy provides possibility,
And determine can occur strong drug action, adverse reaction small characteristic when it is used to treat disease.But siRNA is hydrophilic negatively charged
Macromolecular, limit it and absorbed by histocyte, and is extremely unstable in vivo, easily degraded by RNA enzyme.And it is common
SiRNA transport vehicle such as adenovirus easily causes cell mutation and immune response, and cationic-liposome is to primary cell, immunocyte
Equal transfection efficiencies are low.Therefore, a kind of suitable carrier is found, siRNA is safe and efficient, stable be carried to intracellular is siRNA
Drug moves towards the critical issue of clinical application.Nano particle possesses small-size effect, skin effect and macro quanta tunnel effect
Drug when transport vehicle as drug, can be wrapped in inside due to its special physicochemical properties by equal good characteristics,
It reduces after drug enters in vivo and degradation, hydrolysis and redox reaction occurs, enhance the stability of drug.
Selenium is micro elements needed by human, mainly plays biological function in the form of selenoprotein in vivo and participates in each
Kind physiology link.Selenium and the mechanism of action of selenoprotein participation viral disease occurrence and development are also the research of people's current interest
Hot spot.Research shows that selenium participates in the occurrence and development process of the generations of many virus infections, virulence and viral disease;Host
Intracellular Se content will affect the mutation, duplication and virulence of many intrusive viruses.The missing of selenium can cause some RNA virus bases
Because of a group accumulation for mutation, such as Coxsackie virus B 3, AIDS, influenza A virus, atypical pneumonia coronavirus and Ebola
Virus causes gene structure relevant to virus virulence to change.
There is not been reported for the antiviral study of the siRNA of nanometer selenium delivery at present.
Summary of the invention
An object of the present invention is to provide above-mentioned nanometer selenium supported V P1 gene siRNA, the nanometer selenium supported V P1 base
Because the partial size of siRNA is smaller, immune system can be avoided and reach the smallest capillary, extend the residence time in blood flow;
And its target with higher selectivity, longer half-life period.
The second object of the present invention is to provide the preparation method of above-mentioned nanometer selenium supported V P1 gene siRNA.
The third object of the present invention is to provide the application of above-mentioned nanometer selenium supported V P1 gene siRNA.
An object of the present invention is realized by following methods:A kind of nanometer selenium supported V P1 gene siRNA, the siRNA
The gene loci of interference is the 157th nucleotide;
The siRNA sequence is:
Sense 5 '-ggcaucaucaaaugcuagutt-3 ',
Antisense 5’-acuagcauuugaugaugcctt-3’。
The second object of the present invention is realized by following methods:A kind of nanometer selenium supported V P1 gene siRNA as described above
Preparation method, include the following steps:
Step 1:Vitamin c solution is mixed in bag filter with sodium selenite solution, is stirred, it is saturating to be placed in water progress
Analysis, obtains nanometer selenium solution after purification;
Step 2:Polyethyleneimine (PEI) and siRNA will be added in the nanometer selenium solution, stirring is centrifuged on abandoning
Clearly, by pellet frozen it is dry to get.
Wherein, the concentration of vitamin c solution is 400 μ g/ml in the step 1;The molten concentration of the sodium selenite is 400
μg/ml;The vitamin c solution and the molten volume ratio of sodium selenite are 1:40.
Wherein, the mixing speed of the step 1 is 200-300rpm, mixing time 0.5-1h.The stirring of the step 2
Speed is 200-300rpm, mixing time 0.5-1h.
Wherein, the centrifugal speed of the step 2 is 8000-12000rpm, centrifugation time 15-30min.
Wherein, the mode that the vitamin c solution with sodium selenite solution of the step 1 mix is:By vitamin c solution by
It is added dropwise in sodium selenite solution.
Wherein, the reaction condition of the step 1 is 23 DEG C -27 DEG C.
The third object of the present invention is realized by following methods:A kind of nanometer selenium supported V P1 gene siRNA as described above
Inhibit the drug of EV71 virus infection applied to preparation.
Wherein, the present invention compared with the existing technology, has the following advantages that:
The inhibition site of the siRNA prepared by the present invention for being able to suppress EV71 virus VP 1 gene passes through in the 157th site
PCR and Western blot, which demonstrates the siRNA designed for the site, has greater efficiency to silencing VP1 gene.Wherein PCR is real
The expression of nucleic acid of verifying reality VP1 gene significantly suppresses 62%, i.e. the transfection efficiency of the siRNA is 38%;Western blot
Experiment confirms the siRNA after nanometer selenium delivery transfection, and VP1 expressing quantity is reduced.
SiRNA is hydrophilic electronegative macromolecular, limits it and is absorbed by histocyte, and extremely unstable in vivo
It is fixed, easily degraded by RNA enzyme.And common siRNA transport vehicle such as adenovirus easily causes cell mutation and immune response, it is positive from
Sub- liposome is low to transfection efficiencies such as primary cell, immunocytes.Nano particle possesses small-size effect, skin effect and macro
The good characteristics such as quantum tunneling effect are seen, it, can be by medicine when transport vehicle as drug due to its special physicochemical properties
Object is wrapped in inside, reduces after drug enters in vivo and degradation, hydrolysis and redox reaction occurs, enhance the stability of drug.
Because the partial size of nanometer selenium supported V P1 gene siRNA is smaller, immune system can be avoided and reach the smallest blood capillary
Pipe, and extend the residence time in blood flow;Secondly, nanometer selenium-siRNA carrier enhances the target choosing of siRNA by modification
Selecting property improves the barrier that siRNA overcomes organism, efficiently reaches target spot;Third, the outer layer of nano-carrier is repaired by chemistry
Decorations, improve its solubility and biocompatibility, can avoid the removing of reticuloendothelial system, have longer half-life period, very
SiRNA is discharged to controllability;4th, improve the utilization efficiency of load siRNA.
Detailed description of the invention
Fig. 1 is preparation and the phenogram of nanometer selenium supported V P1 gene siRNA;
Fig. 2 is that nanometer selenium supported V P1 gene siRNA detects figure to Vero cell survival rate;
Fig. 3 is the jamming effectiveness figure that siRNA is transfected with nanometer selenium, PEI;
Fig. 4 is the potential image of SeNPs, Se@PEI and Se@PEI@siRNA;
Fig. 5 is to measure nanometer selenium delivery siRNA to the result figure of Vero cytotoxicity;
Fig. 6 is the interference effect figure for measuring nanometer selenium delivery siRNA.
Specific embodiment
With reference to embodiment, claim of the invention is described in further detail, but do not constituted pair
Any restrictions of the invention, any limited times modification made in the claims in the present invention protection scope, still of the invention
In claims.
Embodiment 1
(1) it is synthesized for the design of EV71 virus VP 1 gene siRNA
The C4 hypotype EV71 virus VP 1 gene area of Asia prevalence is selected, simultaneously chemical synthesis siRNA is designed.
The gene loci of interference is the 157th nucleotide, and siRNA sequence is:
Sense 5 '-ggcaucaucaaaugcuagutt-3 ',
Antisense 5’-acuagcauuugaugaugcctt-3’。
Wherein out-of-order negative control siRNA (scramble siRNA) sequence is:
Sense 5'-gcaagaauggugcacccautt-3';
Antisense 5’-augggugcaccauucuugctt-3’。
(2) preparation of nanometer selenium system and characterization
400 μ g/ml vitamin c solution of 0.1ml is added dropwise in 400 μ g/ml sodium selenite solution of 4ml, magnetic force
Stirring 2 hours, obtains nanometer selenium solution after purification after dialysing.Above-mentioned nanometer selenium solution is taken, PEI and siRNA is added, magnetic force stirs
1h is mixed, the solution 10000rpm after reaction is centrifuged 10 minutes, and deionization is washed three times, and sample is freeze-dried to obtain nanometer selenium delivery
SiRNA nanometer medicine-carried system.
By transmission electron microscope observing shape characteristic after nanometer selenium-siRNA is resuspended with PBS buffer solution, examined by particle size analyzer
It surveys, is as a result detailed in Fig. 1-4, in figure:
A and b is nanometer selenium (SeNPs), the amine-modified nanometer selenium solution (Se PEI) of polyethyleneimine and poly- second respectively in Fig. 1
The transmission electron microscope picture of siRNA (Se@PEI@siRNA) that alkene imines is modified, through nanometer selenium delivery.As can be seen from the figure without
The SeNPs of modification is easy to appear reunion, and the nanometer selenium (i.e. Se@PEI, Se@PEI@siRNA) after functionalization is more equal than SeNPs dispersion
It is even.
Fig. 2 is the Tyndall effect figure of Se@PEI@siRNA.
Fig. 3 is the particle diameter distribution of SeNPs, Se@PEI and Se@PEI@siRNA, as can be seen from the figure SeNPs average grain diameter
For 200nm, Se@PEI average grain diameter is 100nm, and Se@PEI@siRNA average grain diameter is 80nm.The grain of obvious Se@PEI@siRNA
Diameter is smaller, it is easier to enter cell.
Fig. 4 is the Potential distribution of SeNPs, Se@PEI and Se@PEI@siRNA, it can be seen from the figure that the current potential of SeNPs
For -25mv, the current potential of Se@PEI is 6mv, and Se@PEI@siRNA current potential is 12mv.It is analyzed from the data of current potential, Se@PEI@
The absolute value ratio SeNPs of siRNA current potential is big, illustrates that stability is better than SeNPs.
(3) nanometer selenium delivers siRNA to Vero cytotoxicity assay
Cell survival rate is measured with mtt assay:
With 0.25% trypsin digestion cell, with 4 × 104A/ml is inoculated in 96 orifice plates, every 100 μ l of hole.
It is placed in 37 DEG C, 5%CO2After incubator culture for 24 hours, it is separately added into SeNPs, Se@PEI, Se@PEI@siRNA solution
Each 100 μ l continues to cultivate 12h.
Addition 5mg/ml 3- (4,5- dimethylthiazole -2) -2,5- diphenyltetrazolium bromide bromide (MTT), 20 holes μ l/, 37
DEG C be incubated for 5h after inhale abandon cultivation plate hole in liquid, be added dimethyl sulfoxide (DMSO), 150 holes μ l/, vibrate 10min, purple knot
Brilliant object after completely dissolution, measures each hole OD570Value.With control group OD570It is 100%, calculates drug-treated group cell survival rate.Carefully
Born of the same parents' survival rate (%)=(OD570Experimental group/OD570Control group) × 100%.As a result Fig. 5 is detailed in, in figure:
Mtt assay surveys different pharmaceutical component (control group, virus infected cell group, virus infected cell+SeNPs group, virus sense
Contaminate cell Se@PEI, virus infected cell+Se@PEI@siRNA group) to function and effect after cell processing.
After showing virus infected cell, cell survival rate rate is 37%, is decreased obviously relative to control group.Virus infection is thin
After born of the same parents, after difference SeNPs, Se@PEI and Se@PEI@siRNA processing, cell survival rate is respectively 42%, 40%, 73%.More than
After data show virus infected cell, cell survival rate is decreased obviously relative to control group.After virus infected cell, difference Ghana
After the nanometer selenium processing of rice selenium and PEI modification, risen relative to control group.Nanometer selenium delivers siRNA, cell survival rate phase
Other components are obviously risen.
(4) interference effect of nanometer selenium delivery siRNA
Other ingredient processing (EV71 group) are not added before EV71 virus infection for Vero cell, or are separately added into nanometer selenium
SeNPs, SeNPs PEI, negative control siRNA (Se PEI amine-modified through polyethyleneimine and by nanometer selenium delivery
ScramblesiRNA), after Se@PEI@siRNA, 12h, supernatant is abandoned, virus titer is added in PBS after washing 3 times be 100TCID50
EV71 virus liquid be incubated for 2h, renew the fresh DMEM culture medium containing 10% fetal calf serum continue cultivate 48h, extraction cell RNA,
VP1 gene is expanded, Se@PEI@siRNA is calculated to the inhibition efficiency of VP1 gene mRNA expression according to CT value.
As the result is shown:It is detailed in Fig. 6, VP1 gene nucleic acid is expressed as untransfected pair after transfecting Se@PEI@scramble siRNA
According to the 94% of group (EV71 group), as a result without statistical difference;The expression of nucleic acid of VP1 reduces after transfection SeNPs@PEI siRNA
It is 38%, for untransfected control group (EV71 group) compared to there is significant difference, i.e. its jamming effectiveness is 62%.
Enter cell in conclusion the nanometer selenium modified through PEI can effectively deliver siRNA and play efficient interference work
With, and then to Apoptosis caused by viral infection resisting.
Sequence table
<110>Guangzhou Women and Children's Medical Center
<120>Nanometer selenium supported V P1 gene siRNA and the preparation method and application thereof
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA/RNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
ggcaucauca aaugcuagut t 21
<210> 2
<211> 21
<212> DNA/RNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
acuagcauuu gaugaugcct t 21
<210> 3
<211> 21
<212> DNA/RNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
gcaagaaugg ugcacccaut t 21
<210> 4
<211> 21
<212> DNA/RNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
augggugcac cauucuugct t 21
Claims (10)
1. a kind of nanometer selenium supported V P1 gene siRNA, which is characterized in that the gene loci of the siRNA interference is the 157th
Nucleotide;
The siRNA sequence is:
Sense 5 '-ggcaucaucaaaugcuagutt-3 ',
Antisense 5’-acuagcauuugaugaugcctt-3’。
2. a kind of preparation method of nanometer selenium supported V P1 gene siRNA as described in claim 1, which is characterized in that including with
Lower step:
Step 1:Vitamin c solution is mixed in bag filter with sodium selenite solution, is stirred, is placed in water and dialyses, obtain
Nanometer selenium solution after purification;
Step 2:By in the nanometer selenium solution, polyethyleneimine and siRNA is added, both make final concentration be respectively 40 μM and
Supernatant is abandoned in 2.5nM, stirring, centrifugation, by pellet frozen it is dry to get.
3. the preparation method of nanometer selenium supported V P1 gene siRNA according to claim 2, which is characterized in that the step
The concentration of vitamin c solution is 400 μ g/ml in 1.
4. the preparation method of nanometer selenium supported V P1 gene siRNA according to claim 2, which is characterized in that the step
The molten concentration of sodium selenite is 400 μ g/ml in 1.
5. the preparation method of nanometer selenium supported V P1 gene siRNA according to claim 3, which is characterized in that the step
Vitamin c solution and the molten volume ratio of sodium selenite are 1 in 1:40.
6. the preparation method of nanometer selenium supported V P1 gene siRNA according to claim 2, which is characterized in that the step
1 mixing speed is 200-300rpm, mixing time 0.5-1h.
7. the preparation method of nanometer selenium supported V P1 gene siRNA according to claim 2, which is characterized in that the step
2 mixing speed is 200-300rpm, mixing time 0.5-1h.
8. the preparation method of nanometer selenium supported V P1 gene siRNA according to claim 2, which is characterized in that the step
2 centrifugal speed is 8000-12000rpm, centrifugation time 15-30min.
9. the preparation method of nanometer selenium supported V P1 gene siRNA according to claim 2, which is characterized in that the step
The mode that 1 vitamin c solution is mixed with sodium selenite solution is:Vitamin c solution is added dropwise to sodium selenite solution
In.
10. a kind of nanometer selenium supported V P1 gene siRNA as described in claim 1, which is applied to preparation, inhibits EV71 virus infection
Drug.
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YINGHUA LI等: "Delivery of VP1 siRNA to inhibit the EV71 virus using functionalized silver nanoparticles through ROSmediated signaling pathways", 《THE ROYAL SOCIETY OF CHEMISTRY》 * |
YINGHUA LI等: "Multifunctional selenium nanoparticles as carriers of HSP70 siRNA to induce apoptosis of HepG2 cells", 《INTERNATIONAL JOURNAL OF NANOMEDICINE》 * |
ZHENGFANG LIN等: "Inhibition of Enterovirus 71 by Selenium Nanoparticles Loaded with siRNA through Bax Signaling Pathways", 《AMERICAN CHEMICAL SOCIETY》 * |
吕玉春等: "靶向肠道病毒71型VP1基因的siRNA表达质粒的构建 ", 《中国生物制品学杂志》 * |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2023082213A1 (en) * | 2021-11-13 | 2023-05-19 | 暨南大学 | Novel coronavirus protein antigen nanovaccine, preparation method therefor and application thereof. |
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