CN100422326C - Si RNA capable of controlling foot-and-mouth disease virus copying and infection and its preparing method and use - Google Patents
Si RNA capable of controlling foot-and-mouth disease virus copying and infection and its preparing method and use Download PDFInfo
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- CN100422326C CN100422326C CNB200410054028XA CN200410054028A CN100422326C CN 100422326 C CN100422326 C CN 100422326C CN B200410054028X A CNB200410054028X A CN B200410054028XA CN 200410054028 A CN200410054028 A CN 200410054028A CN 100422326 C CN100422326 C CN 100422326C
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Abstract
The present invention relates to siRNA capable of inhibiting foot-and-mouth disease virus (FMDV) from being replicated and infected in animal cells and individuals, and a preparation method and application thereof. Highly conservative sequence parts in five sections namely 5NCR, VP4, VPg, POL and 3NCR of an FMDV genome sequence are selected as target sequences of siRNA which is designed by any continuous sequence of 21 to 25 bp in the target sequences. Cell toxicity attack experiments prove that the siRNA capable of complementing and pairing with the DNA sequences of the target genes can effectively inhibit the replication and the infection of different 0 type FMDV toxic strains in BHK21 cells and can effectively protect neonate rats against resisting virus infection and have favorable protective action on cells and neonate rats by using highly conservative sequences in FMDV genomes as target genes.
Description
Technical field
The invention belongs to foot and mouth disease virus (FMDV) Prevention Technique field, be specifically related to genomic conserved sequence at FMDV, utilize in-vitro transcription to obtain siRNA or chemosynthesis obtains siRNA, or plasmid vector expresses siRNA in bacterial cell, and this siRNA can suppress the infection that FMDV duplicated and prevented and treated FMDV on the cell levels or on the animal individual level.
Background technology
Livestock foot-and-mouth disease is cloven-hoofed animals such as livestock contagious disease the most serious in the world today, main harm pig, ox, sheep, and World Organization for Animal Health classifies foot and mouth disease first of the category-A transmissible disease as.For many years, foot and mouth disease is large-scale outbreak and popular worldwide, causes the tremendous economic loss to livestock industry.According to immunogenicity, foot and mouth disease virus has A, O, C, SATI, SATII, SATIII and Asia I totally 7 serotype and the hypotype more than 65.There is not cross-immune reaction between foot and mouth disease virus (FMDV) is various; one type vaccine can not protect domestic animal to avoid the infection of another kind of class C-type virus C; have been found that to have an above viral prevalence in many areas, if, bear the character of much blindness with the vaccine immunity domestic animal of a type.In order to overcome the limitation of this immunity, the preparation that research has the effect of wide spectrum foot-and-mouth disease virus resistant has important practice significance.In addition, because FMDV's has a quick infectivity and pathogenic by force, control the popular obstacle that brought of foot and mouth disease fast for the traditional vaccine of use.
The RNAi technology is one of significant achievement of obtaining in recent years of scientific research field, and its biggest advantage is to have the validity of height and specificity and has defence and result of treatment fast.Its treatment field that acts on gene functional research field and the various diseases especially treatment field of virus disease has shown immeasurable value, for example at anti-AIDS virus (HIV), hepatitis C virus (HCV), find all in the research of hepatitis B virus (HBV) and poliomyelitis virus virus diseases such as (Poliovirus) that RNA disturbs, fabulous for the print effect that suppresses this viroid, may found new, more effective approach for the treatment of this viroid disease.But at occurring in nature, all can there be different mutant strains in various viruses, and foot and mouth disease virus just has seven kinds of serotypes and a lot of variants.This has brought huge difficulty for the preventing and controlling of hoof-and-mouth disease viral disease.Because siRNA disturbs the height specificity of target sequence, makes the RNAi technology also run into inevitable obstacle in the application prospect of prevention and cure of viruses.
Summary of the invention
The objective of the invention is to propose a kind of energy and suppress the siRNA that the even not isostructural foot and mouth disease virus of different strains is duplicated and infects in the same type in zooblast and individuality, and the preparation method of this siRNA and application.
What the present invention proposed can suppress the siRNA that FMDV duplicates and infects, be to classify benchmark as with the genome sequence of each type, each hypotype, different strains, choose the conservative 5NCR of FMDV camber, VP4, VPg, POL, the 3NCR (see figure 1) is a target sequence, design obtains siRNAs, and the siRNA that obtains can disturb duplicating of different strains and veriform FMDV in the same type and infect.
Five kinds have the gene of homology or the target sequence that section all can be used as siRNA to each type and hypotype and different strains among the present invention.
Among the present invention, the RNA sequence of any successive 21-25bp length all can be prepared as siRNA in the above-mentioned selected target sequence, and this siRNA all has the effect of disturbing FMDV to duplicate and infect.
SiRNA of the present invention can prepare with following method: external synthetic, transcribe the back and obtain the siRNAs mixture, also available plasmid vector expression in vivo or directly synthesize siRNAs. at any one section 21-25bp of target sequence by chemical synthesis with the Dicer enzymic digestion
Conserved regions among the 5NCR that is adopted among the present invention is 149bp (500-648 position Nt), and its sequence is SEQ IDNO1; Conserved regions among the VP4 is 170bp (1652-1821 position Nt), and its sequence is SEQ ID NO2; Conserved regions among the VPg is 137bp (5798-5934 position Nt), and its sequence is SEQ ID NO3; Conserved regions among the POL is 185bp (7694-7878 position Nt), and its sequence is SEQ ID NO4; Conserved regions among the 3NCR is 91bp (8012-8102 position Nt), and its sequence is SEQ ID NO5.
After obtaining these sequences by the RT-PCR method from viral genome, add that by PCR method the T7 promoter sequence is to obtain utilizing t7 rna polymerase to carry out the template DNA of in-vitro transcription respectively.Recombinate behind the dsRNA that the Dicer enzymic digestion transcribes through in-vitro transcription and people, obtain the mixture (Fig. 2) of the siRNA of 21-25bp.This siRNA mixture has comprised the siRNA of the 21-25bp of the various combined sequence in conservative section.On cell levels, carry out the interference experiment that FMDV duplicates and infects with the siRNA mixture, obtained the interference effect (Fig. 3) of 82-99%.
Use earlier the virus attack cell, the mode of transfection siRNA of the present invention suppresses FMDV and duplicates and infect again, also can obtain good protection effect, and protection efficient is attacked malicious mode after being higher than first transfection.
The present invention has chosen the conservative section of FMDV genome camber as target sequence, realized the interference effect of duplicating Yu infecting of siRNA to different strains, the prevention and the treatment that are used for hoof-and-mouth disease viral diseases such as domestic animal, pig, ox, sheep for the RNAi technology provide new technological line.
Description of drawings
Fig. 1: selected conservative section synoptic diagram among the present invention.Be specially the target gene of foot-and-mouth disease virus gene group and corresponding siRNA.The target fragment of selecting is named with protogene, and goes out with the thick line segment mark, the numeral of target sequence be the position of Nucleotide in HKN/2002 (GeneBankAccessionAY317098.1) strain.
Fig. 2: the siRNA mixture synoptic diagram that obtains among the present invention.The siRNAs that from dsRNA, produces.
Fig. 3: the siRNA specificity suppresses target gene expression in the BHK21 cell.A wherein, reporter plasmid and corresponding or incoherent siRNA cotransfection cell, the representational photo of taking pictures and obtaining with fluorescent microscope after 24 hours; B, siRNA is to the inhibition of EGFP expression activity; C, the RT-PCR method is estimated the inhibition of siRNA to the EGFP expression activity.
Fig. 4: siRNA has reduced the virus titer of virus in the BHK21 cell.A wherein, the siRNA specificity suppresses HKN/2002 virus duplicating in the BHK21 cell; B, the siRNA specificity suppresses CHA/99 virus duplicating in the BHK21 cell; C, siRNA can not specificity suppress Pseudorabies virus (PRV) duplicating in the BHK21 cell.
Fig. 5: the siRNA of two kinds of experiment methods compares the inhibition effect of virus replication in the BHK21 cell.
Fig. 6: the quick antiviral response that synthetic siRNA induces suckling mouse in the body, improve the survival rate after suckling mouse is subjected to virus attack.
Embodiment
With 5NCR, VP4, VPg, POL, 3NCR is that the target area section specifically describes the present invention.With RT-PCR single stage method from the cell culture fluid of FMDV O type strain HKN/2002 strain, increase respectively above-mentioned 5 target area sections, i.e. 5NCR (SEQ ID NO.1), VP4 (SEQ ID NO.2), VPg (SEQ ID NO.3), POL (SEQ ID NO.4), 3NCR (SEQ ID NO.5).The sequence of each section is all determined through dna sequencing.
In order to analyze the interference effect of siRNA to each target area section, we have made up a series of reporter plasmids, be about to 5 target area sections and be cloned into 5 ' end of a fluorescence protein expression carrier (pEGFP-N1) respectively, constitute fusion gene (as 5NCR-EGFP), this gene enters cell by the reporter plasmid transfection, can express the fluorescin (EGFP) of fusion in cell.The expression amount of EGFP reduces if the target area section is disturbed then by siRNA, can judge in view of the above whether specificity is disturbed target gene to siRNA.
In order to obtain the siRNA of each section by in-vitro transcription and enzyme blanking method, at first the T7 promoter sequence is added on 5 target area sections by PCR method, obtain transcribing template, carry out in-vitro transcription with t7 rna polymerase then and obtain long dsrna (dsRNA).With the Dicer enzymic digestion dsRNA of reorganization, obtain the siRNA mixture (accompanying drawing 2) of short about 22bp after purified.
At first we are with the reporter plasmid cotransfection BHK21 cell of 100ng siRNA and 200ng, after cultivating 24h, expression (Fig. 3 A) with fluorescence microscope EGFP, the siRNA that the result shows 5 sections has tangible interference effect to target area Duan Jun separately, and the expression jamming effectiveness of EGFP is reached 84%-99% (Fig. 3 B); MRNA mensuration (RT-PCR) by fusion gene in the pair cell proves that also target gene expression is suppressed (Fig. 3 C) equally.
For further checking siRNA is to the interference performance of virus replication, we have carried out the virus replication interference experiment of cell levels.A kind of mode is to use the siRNA transfection BHK21 cell of 100ng earlier, uses 100TCID behind the 5h
50The HKN/2002 of dosage virus liquid inductance transfect cell, behind the 24h with inverted microscope observation of cell pathology situation, and in 48h and 96h respectively the collecting cell nutrient solution to determine the interference performance of siRNA by measuring virus titer.Attack poison to detect the cross protection effect of siRNA with another FMDV strain CHA/99 simultaneously.Another kind of mode is to use 100TCID earlier
50The HKN/2002 virus liquid inductance transfect cell of dosage, the siRNA transfection BHK21 cell of usefulness 100ng behind the 1h, and in 48h and 96h difference collecting cell nutrient solution mensuration virus titer.The result shows; the siRNA of 5 sections all can effectively suppress duplicating of virus; make cell avoid cracking (Fig. 4 A); and the strain of different sources there is cross-protection (Fig. 4 B); but xenogenesis virus is not had restraining effect fully as Pseudorabies virus (PRV), and this shows that designed siRNA has sequence-specific (Fig. 4 C).Even the experimental result of another kind of mode shows virus and infect FMDV earlier, siRNA still can suppress duplicating of virus, prevents the cytopathy cracking, and interference effect is than the first transfection siRNA experimental group more obvious (Fig. 5) of infective virus again.
Then, we design siRNA with the Nucleotide of one section 21NT in the POL section and 63NT for target, and are designed to be expressed as in vivo the hairpin structure of siRNA with the plasmid vector form.With this construction through the subcutaneous immune suckling mouse of neck and use 100LD
50HKN/2002 virus attack suckling mouse.Compare with control group (with physiological saline immunity or incoherent siRNA plasmid construction thing pNTH), experimental group (pNT21 and p63NT) can make 80% suckling mouse survival more than 5 days (Fig. 6).
These presentation of results, the infection that can on cell levels and animal individual level, disturb FMDV at the siRNA of conserved regions design with duplicate, therefore, can be used as the preparation of effective prevention and treatment FMDV.
SEQUENCE?LISTING
＜110〉Fudan University
＜120〉a kind ofly can suppress foot and mouth disease virus and duplicate and siRNA of infecting and its production and application
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Claims (3)
1. one kind can be suppressed the siRNA that FMDV duplicates and infects, it is characterized in that sequence with one of the following high conservative in the FMDV genome is as its target sequence, by PCR method and interpolation T7 promoter sequence, obtain to utilize t7 rna polymerase to carry out the template DNA of in-vitro transcription; Through the recombinate mixture of siRNA of any successive 21-25bp behind the dsRNA that the Dicer enzymic digestion transcribes of in-vitro transcription and people; These sequences are respectively SEQ ID NO1:5NCR, SEQ ID NO2:VP4, SEQ ID NO3:VPg, SEQ ID NO4:POL, SEQ ID NO5:3NCR.
2. the FMDV that can suppress according to claim 1 duplicates preparation method with the siRNA that infects, it is characterized in that each sequence respectively by PCR method and add the T7 promoter sequence, obtain to utilize t7 rna polymerase to carry out the template DNA of in-vitro transcription; Recombinate behind the dsRNA that the Dicer enzymic digestion transcribes through in-vitro transcription and people, obtain the mixture of the siRNA of any successive 21-25bp; Described each sequence is respectively SEQ ID NO1:5NCR, SEQ IDNO2:VP4, SEQ ID NO3:VPg, SEQ ID NO4:POL, SEQ ID NO5:3NCR.
A siRNA as claimed in claim 1 preparation suppress FMDV duplicate with the preparation that infects in application.
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| CNB200410054028XA CN100422326C (en) | 2004-08-26 | 2004-08-26 | Si RNA capable of controlling foot-and-mouth disease virus copying and infection and its preparing method and use |
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| CNB200410054028XA CN100422326C (en) | 2004-08-26 | 2004-08-26 | Si RNA capable of controlling foot-and-mouth disease virus copying and infection and its preparing method and use |
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| CN100422326C true CN100422326C (en) | 2008-10-01 |
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| CN100345965C (en) * | 2005-08-04 | 2007-10-31 | 复旦大学 | siRNA capable of inhibiting foot and mouth disease virus replication and infection and its preparation method |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1408434A (en) * | 2002-09-16 | 2003-04-09 | 复旦大学 | Polypeptide and DNA combined vaccine for resisting animal's foot-and-mouth disease and its preparing method |
| CN1465584A (en) * | 2003-07-09 | 2004-01-07 | 北京交通大学 | SiRNA target sequence bank of SARS virus and use thereof |
| CN1470285A (en) * | 2003-06-13 | 2004-01-28 | 复旦大学 | Polypeptide vaccine of anti Asiatic I virus of foot-and-mouth disease and its preparing method |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1408434A (en) * | 2002-09-16 | 2003-04-09 | 复旦大学 | Polypeptide and DNA combined vaccine for resisting animal's foot-and-mouth disease and its preparing method |
| CN1470285A (en) * | 2003-06-13 | 2004-01-28 | 复旦大学 | Polypeptide vaccine of anti Asiatic I virus of foot-and-mouth disease and its preparing method |
| CN1465584A (en) * | 2003-07-09 | 2004-01-07 | 北京交通大学 | SiRNA target sequence bank of SARS virus and use thereof |
Non-Patent Citations (2)
| Title |
|---|
| SIRNA 的设计,合成与转染及其在RNA干扰中的应用. 国外医学临床生物化学与检验学分册，第24卷第4期. 2003 |
| SIRNA 的设计,合成与转染及其在RNA干扰中的应用. 国外医学临床生物化学与检验学分册，第24卷第4期. 2003 * |
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