CN103642808A - Three siRNAs (small interfering Ribose Nucleic Acids) interfering with 2A protease gene of EV71 virus and application thereof - Google Patents
Three siRNAs (small interfering Ribose Nucleic Acids) interfering with 2A protease gene of EV71 virus and application thereof Download PDFInfo
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- CN103642808A CN103642808A CN201310646161.3A CN201310646161A CN103642808A CN 103642808 A CN103642808 A CN 103642808A CN 201310646161 A CN201310646161 A CN 201310646161A CN 103642808 A CN103642808 A CN 103642808A
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Abstract
The invention discloses three siRNAs (small interfering Ribose Nucleic Acids) interfering with the 2A protease gene of EV71 virus and an application thereof. Stealth siRNA-69, stealth siRNA-294 and stealth siRNA-319 are three siRNAs which are chemically synthesized with the 2A protease gene of EV71 as the target sequence; the three siRNAs can be effectively and complementarily combined with the 2A protease gene regions of the EV71 virus to degrade the nucleic acid of the virus; therefore, the purpose of inhibiting virus replication and amplification is achieved. RNAi (Ribose Nucleic Acid interfere) based on the siRNAs opens up a new way for clinical treatment of virus infection type diseases due to high specificity and high efficiency of gene silencing thereof.
Description
Technical field
What the present invention relates to is three siRNA and the application thereof of disturbing EV71 virus 2A proteinase gene, comprises stealth siRNA-69stealth siRNA-294stealth siRNA-319.
Background technology
EV71 virus belongs to picornaviridae enterovirus A group, and viral genome is sub-thread positive chain RNA, big or small about 7500bp, and viral capsid is the three-dimensional symmetrical globosity of icosahedron, diameter is about 24-30nm, without coating and outstanding.EV71 infects and mainly causes hand foot mouth disease, multiplely be born in preschool children, its clinical symptom main manifestations is heating, there is bleb in the positions such as hand, foot, oral mucosa, there is the symptoms such as meningitis, encephalitis, encephalomyelitis, pulmonary edema and cycle penalty in morbidity after 1-5 days in severe cases, even dead.Nineteen fifty-seven, Canadian first report hand foot mouth disease, is very popular in succession in Europe, a plurality of areas in America and Asia afterwards.In China, hand foot mouth disease early has popular.1981, Shanghai first report hand foot mouth disease, in March, 2008, there is fairly large epidemic situation in Anhui Province's Fuyang City, number of the infected reaches 176000 people, and have 23 examples dead, in other areas, the whole nation, also there is immediately the epidemic situation of hand foot mouth disease, caused social fear.At present, there is no the antiviral of effective opposing EV71, so it is very necessary finding a kind of potential effective antiviral method.
It is the gene silencing phenomenon of being brought out by double-stranded RNA on a kind of molecular biology that RNA disturbs, and its mechanism is by hindering the translation of specific gene or transcribing inhibition of gene expression.While importing the double-stranded RNA with endogenous mRNA coding region homology in cell, there is degraded and cause genetic expression reticent in this mRNA.The double stranded rna molecule importing in research is the double-stranded small RNA molecular (being called siRNA) with the about 21-25 Nucleotide of size of gene silencing function, in RNAaseIII family, double-stranded RNA is had to specific enzyme Dicer and processes.
Reported in recent years that other regions with EV71 gene were such as vp1, vp2,5 ' UTR, 3 ' UTR, 2C, 3Cpro and3Dpol is that target sequence chemosynthesis siRNR treats viral article, but does not also using 2A proteinase gene as the target that suppresses virus replication, so the object of the invention is to inquire into EV712A protease-based because whether three siRNA of target sequence chemosynthesis have potential antivirus action.
Therefore, there is defect in prior art, needs to improve.
Summary of the invention
Technical problem to be solved by this invention is to provide for the deficiencies in the prior art three siRNA and the application thereof of disturbing EV71 virus 2A proteinase gene.
Technical scheme of the present invention is as follows:
Three siRNA that disturb EV71 virus 2A proteinase gene, comprise three siRNA:stealth siRNA-69, stealth siRNA-294, stealth siRNA-319 as shown in the table:
The application of three described siRNA, the effective complementary combination in 2A proteinase gene region of itself and EV71 virus, by viral nucleic acid degraded, thereby reaches the object that suppresses virus replication amplification.
Stealth siRNA-69, stealth siRNA-294, stealth siRNA-319 is with EV712A protease-based because three siRNA of target sequence chemosynthesis, its can with the effective complementary combination in 2A proteinase gene region of EV71 virus, by viral nucleic acid degraded, thereby reach the object that suppresses virus replication amplification.Take siRNA as high degree of specificity and the high efficiency of basic RNAi because of its gene silencing, for the clinical treatment of virus infection class disease has been opened up new approach.
Accompanying drawing explanation
Fig. 1 is the detected result of stealth siRNA antivirus action; Stealth siRNA suppresses the out of order siRNA of viral RNA as negative control; Simulation transfection treatment group is for only processing with Lipo2000; Only virus is for only using virus infection.Three times independent experiment data represent (* * p<0.01) with means ± SD.
Fig. 2 is that stealth siRNA suppresses viral protein expression; Actin contrasts as internal reference.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in detail.
1stealth siRNA's is synthetic
In NCBI site search EV712A proteinase gene sequence (JN001860.1), take this sequence as target is by three siRNA of Shanghai Ying Jun company chemosynthesis, its action principle is that stealth siRNA transfection is entered after cell, thereby in conjunction with a ribozyme mixture, form the reticent mixture (RISC) of RNA induction, double-stranded stealth siRNA is broken down into strand and forms the RISC activating under the effect of enzyme subsequently, the RISC activating navigates on the mRNA of homology by base pairing, and at the position cutting said target mrna apart from 12 bases of siRNA3 ' end, thereby suppress the translation of mRNA.Its sequence sees the following form 1.
The correlated series of three stealth siRNA molecules of table 1 and its site at 2A proteinase gene
2stealth siRNA transfection RD cell and virus infection
2.1 transfection the day before yesterdays, by RD cell kind in 24 porocyte culture plates, every hole 400 μ l cell suspensions, density is 0.5X10
5cells/ hole, each experimental group is established three parallel holes, and in substratum, not containing microbiotic, 37 spend 5%CO
2spend the night, during transfection, cell density reaches 50% left and right.
2.2 3 Stealth siRNAs be take respectively concentration that final concentration is 60nM and are dissolved in 50 μ l OPTI-MEM substratum and mix dilution, standing 5 minutes of room temperature.
2.3 are dissolved in 2 μ l Lipofeetamine2000 in 50 μ l OPTI-MEM substratum and mix dilution, standing 5 minutes of room temperature.
2.4 mix above-mentioned two step mixed solutions, and room temperature adds in 24 orifice plates after standing 20 minutes, is placed in 37 degree, 5%CO
2cell culture incubator is cultivated.
After 2.56h by transfection in siRNA hole substratum with replacing containing the substratum of 2%FBS, be placed in cell culture incubator and continue to cultivate.
The EV71 virus infected cell that is 0.01 by infection multiplicity after 2.6 transfection 24h.
The detection of 3stealth siRNA antivirus action
In order to detect three stealth siRNAs to viral restraining effect, we adopt real-time RT-PCR and western blot respectively viral RNA and viral capsid proteins vp1 to be detected.As illustrated in fig. 1 and 2, the amount of the viral RNA of three siRNA treatment group and vp1 albumen obviously reduce, and stealth siRNA-69 to suppress viral ability the strongest, stealth siRNA-319 is the most weak.
Take siRNA as high degree of specificity and the high efficiency of basic RNAi because of its gene silencing, for the clinical treatment of virus infection class disease has been opened up new approach.
Should be understood that, for those of ordinary skills, can be improved according to the above description or convert, and all these improvement and conversion all should belong to the protection domain of claims of the present invention.
Claims (2)
1. three siRNA that disturb EV71 virus 2A proteinase gene, is characterized in that, comprise three siRNA:stealth siRNA-69, stealth siRNA-294, stealth siRNA-319 as shown in the table:
2. the application of three siRNA claimed in claim 1, the effective complementary combination in 2A proteinase gene region of itself and EV71 virus, by viral nucleic acid degraded, thereby reaches the object that suppresses virus replication amplification.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102337263A (en) * | 2010-07-21 | 2012-02-01 | 苏州瑞博生物技术有限公司 | siRNA (Small interfering ribonucleic acid) capable of inhibiting expression of enterovirus 71 type gene, composition and application |
| CN103382472A (en) * | 2012-05-23 | 2013-11-06 | 中国人民解放军第四军医大学 | New targets for interference of EV71, small interfering RNAs and application thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102337263A (en) * | 2010-07-21 | 2012-02-01 | 苏州瑞博生物技术有限公司 | siRNA (Small interfering ribonucleic acid) capable of inhibiting expression of enterovirus 71 type gene, composition and application |
| CN103382472A (en) * | 2012-05-23 | 2013-11-06 | 中国人民解放军第四军医大学 | New targets for interference of EV71, small interfering RNAs and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| GU ET AL.: "JN001860.1", 《GENBANK》 * |
| 刘海冰等: "干扰EV71病毒2Apro基因的siRNA具有潜在的抗病毒作用", 《江苏大学学报（医学版）》 * |
| 王胜军等: "干扰EV71病毒2A蛋白酶基因的siRNA具有潜在的抗病毒作用", 《中华医学会第十次全国临床微生物学术年会暨第九届全球华人临床微生物与感染症学术论坛暨2013年浙江省医学微生物与免疫学及医学病毒学学术年会论文汇编》 * |
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Application publication date: 20140319 |