CN102154290B - SiRNAs for inhibiting epidemic encephalitis B viruses - Google Patents
SiRNAs for inhibiting epidemic encephalitis B viruses Download PDFInfo
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- CN102154290B CN102154290B CN201110005104.8A CN201110005104A CN102154290B CN 102154290 B CN102154290 B CN 102154290B CN 201110005104 A CN201110005104 A CN 201110005104A CN 102154290 B CN102154290 B CN 102154290B
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Abstract
The invention relates to siRNAs for inhibiting epidemic encephalitis B viruses and a design method and use thereof. Particularly, based on the whole genome sequence of the epidemic encephalitis B, conserved sequences of separated strains of a genotype 1, a genotype 2, a genotype 3 and a genotype 4 are selected as target sequences. After the siRNAs designed according to the target sequences are transferred into cells, the expression of the genes of the encephalitis B viruses can be inhibited effectively; and when different siRNA molecules are connected in series and transferred into cells, a better inhibition effect can be obtained. The invention also relates to the use of the siRNA molecules in the inhibition of the expression of the gene of the epidemic encephalitis B viruses and in the preparation of medicines for treating and/or preventing epidemic encephalitis B. The invention provides a new approach for inhibiting infection caused by encephalitis B viruses of different genotypes.
Description
Technical field
The present invention relates to the siRNA (siRNA) that can induce RNA to disturb, particularly relate to the siRNA that can suppress epidemic encephalitis B virus, and method of design and purposes.
Background technology
Epidemic encephalitis B virus is called for short encephalitis b virus, is the infecting both domestic animals and human acute infectious disease through killing propagation, is one of the most serious cause of disease causing in the world people's encephalitis.1871, Japan's reported first this disease popular, nineteen thirty-five, Japanese scholars was separated to this virus from the encephalitis patient's that dies of illness cerebral tissue, so in the world by this pathogenic agent called after japanese encephalitis virus (Japanese encephalitisvirus, JEV).According to WHO statistics, global every annual 50000 examples of falling ill, dead 15000 examples.About 1/3rd patient can be therefore and dead, has the patient of neural system sequela to account for 40%-70% in the patient of survival, this disease Major Epidemic in Asia, about more than 20 country in south east asia.Since the nineties in 20th century, the epidemic regions of encephalitis constantly expands, nineteen ninety-five Papua New Guinea and Australian northern island occur the eruption and prevalence of encephalitis in Australian native country, occurring encephalitis case in 1998.Encephalitis in 2005 is in India and Nepal's eruption and prevalence, and thousands is ill.China region is wide, accounts for 1/4th regions, Asia, and the encephalitis morbidity number of China accounts for the whole world the more than 80% of number of always falling ill, and is the popular big country of encephalitis.According to the law on the prevention and control of infectious diseases > > of the < < People's Republic of China (PRC), epidemic encephalitis type B belongs to Category B notifiable disease.From 1938, by Serologic test, confirm that encephalitis is popular and be separated to after the first strain encephalitis b virus in Beijing in 1940 in China, encephalitis repeatedly breaks out within the border in China.Nearly ten years, although the morbidity number of encephalitis is the trend of reduced year by year, be not also very popular, still have every year 20000-30,000 case load.
Encephalitis B is also a kind of Natur al foca transmissible disease of infecting both domestic animals and human simultaneously, and in animal, pig is this sick main harm object.It is reported that the sickness rate of swinery is generally 20-30%, the stillborn foetus of first farrowing sow and mummy ratio can reach 50% left and right.Therefore the Main Economic loss that Gai Bing infected pigs causes is farrowing sow generation breeding difficulty, the healthy and stable development of serious harm pig industry.
For encephalitis B, there is no special treatment measure, vaccination is still the most effective preventive measures of protection Susceptible population.Although vaccination can be protected not infection population effectively, the encephalitis patient for having infected, lacks strong treatment means, so the methods for the treatment of method of application modern means of science and technology development inhibition infection and encephalitis B virus infection easily and effectively just seems particularly important.
Encephalitis b virus belongs to flaviviridae in classification, Flavivirus, and its genome is sub-thread positive chain RNA molecule, is about 11kb, molecular weight is about 4 * 10
6dalton, settling ratio is 42s.Whole genome is by 5 ' end non-translational region (5 '-untranslated regions, UTR), almost crossing over whole genomic single open reading frame (open reading frame, ORF) and 3 ' for one holds non-translational region (3 '-UTR) to form.5 ' 95 Nucleotide of end and 586 Nucleotide of 3 ' end are non-coding region, from 5 ' end to 3 ' gene order of end coding is three structural protein, comprises capsid protein C (capsid), membranin M (membrane, PrM is precursor) and envelope protein E (envelope), seven non-structural protein NS 1s, NS2A, NS2B, NS3, NS4A, NS4B and NS5, approximately 3430 amino acid of encoding altogether, its genome structure is shown in Fig. 1.According to the genomic nucleotide sequence otherness of encephalitis b virus, the encephalitis b virus strain of having identified can be divided into the genotype that four definite genotype and another one are inferred.Gene 1 type and gene 3 types are mainly distributed in most of Asian countries such as China, Korea S, India, Thailand, Philippines, are main popular genotype.Gene 2 types and gene 4 types are mainly distributed in the countries such as Malaysia, Indonesia and north Australia, are endemic genotype.
RNA disturbs (RNA interfering, RNAi) technology be new development in recent years get up a kind of efficiently, the Gene silence of high specificity, be a kind of by the mediation of double-stranded RNA (dsRNA) or microRNA (microRNA, miRNA), transcribe rear mRNA level and close the sequence-specific gene silencing mechanism that corresponding gene is expressed.It is also a kind of protection mechanism that vivo gene group is resisted external infection and inner swivel base, is extensively present in various biologies, as plant, fungi, insect, metazoan and Mammals, comprises the mankind.Concerning external source dsRNAs, in dsRNAs is imported into body after, dsRNA is cut into the siRNA s (small interfering RNAs, siRNAs) of 21-25nt by Dicer.These siRNAs are in conjunction with the target mRNAs of homology in specificity degraded sequence after RNA dependency silencing complex (RISC).For endogenous miRNAs, source transcript (the primary RNAtranscripts of RNA, pri-miRNAs) in nucleus, by Drosha, cut into miRNA precursor (the miRNA precursors of 60-70nt, pre-miRNAs) after, by Exportin-5, be transported in tenuigenin, in tenuigenin, pre-miRNAs is cut into ripe miRNAs by Dicer.Ripe miRNAs exercises cutting target mRNA or suppresses the effect of translation.Most pri-miRNA transcript is all arranged under rna plymerase ii promotor with the form of cluster, with polycistronic form, expresses a plurality of different miRNAs.In mammalian cell, external source imports siRNAs or ShorthairpinRNA (short hairpin RNA, the shRNA) expression vector of the 21-29nt of chemosynthesis all can induce RNAi.Its treatment field that acts on gene functional research field and the various diseases especially treatment field of virus disease has shown immeasurable value.
By RNAi technology, select different target site application siRNA to suppress virus replication and infect open in multiple virus.For encephalitis b virus, research in the past has been reported and can effectively suppress siRNAs or the shRNAs that encephalitis b virus copies.But the siRNAs of these reports or shRNAs are all only for a certain strain encephalitis b virus in gene 3 types, this characteristic makes its effect be subject to great restriction, because, polytropy due to encephalitis b virus geneome RNA sequence, for the effective siRNA of a certain strain virus in gene three types, be difficult to guarantee in other strain in gene 3 types effective, enable to be suitable in gene 3 types, be also difficult to guarantee that it can be suitable in other three kinds of genotype.In addition, can there is the sudden change of escaping in the position of its target sequence in the virus under RNAi suppresses for a long time, and single or two siRNAs are easy to lose effect due to the generation of the sudden change of escaping.So the means that the most effectively suppress virus replication are the drug combinations for many siRNAs of encephalitis b virus conserved regions target sequence.And this scheme was not relating in the design for the siRNAs of encephalitis b virus in the past.
Summary of the invention
The present invention's application RNAi technology, carries out the inhibition of encephalitis b virus and studies, and verified the validity that a plurality of siRNA apply simultaneously by the siRNA for encephalitis b virus genome conserved regions.Particularly, the present invention includes the following aspects:
One aspect of the present invention relates to the molecule that induction RNA disturbs, and it comprises positive-sense strand and antisense strand, it is characterized in that described positive-sense strand or antisense strand are selected from the sequence of following (1)-(5):
(1) arbitrary sequence shown in SEQ ID NO:1～SEQ ID NO:9 and SEQ ID NO:33～SEQ IDNO:50;
(2) with SEQ ID NO:1～SEQ ID NO:9, SEQ ID NO:33～SEQ IDNO:50 in the identity of arbitrary sequence be at least 70% sequence;
(3) through the sequence in (1) or (2) that is selected from of transformation, it is to be added with the sequence of 60 Nucleotide at the most in 5 ' direction of described sequence and 3 ' direction, 5 ' direction or 3 ' direction;
(4) sequence of the sequence that is selected from (1)-(3) operationally being connected and being obtained according to 5 '-3 ' direction, wherein operationally the sequence of series connection is a kind, its copy number is more than 2 or 2; With
(5) sequence of the sequence that is selected from (1)-(3) operationally being connected and being obtained according to 5 '-3 ' direction, wherein operationally the sequence of series connection is two or more, the copy number of every a kind is at least 1.
Wherein the identity of (2) preferably at least 80%, and more preferably at least 84%, more preferably at least 89%, more preferably at least 94%.
The Nucleotide number of wherein adding in (3) is 50 at the most, preferably at the most 40, and more preferably at the most 30, more preferably at the most 20, most preferably at the most 10.
Wherein (4) described sequence is the sequence that the sequence of SEQ ID NO:2, the SEQID NO:34 of 3,6 or 9 copies or SEQ ID NO:43 is operationally connected and obtained, for example, be sequence shown in SEQID NO:51～SEQ ID NO:53; Wherein (5) described sequence is the sequence that the sequence of SEQ ID NO:1～4, SEQ ID NO:5～9, SEQ ID NO:1～9, SEQ ID NO:33～36, SEQ IDNO:37～41, SEQ ID NO:33～41, SEQ ID NO:42～45, SEQ ID NO:46～50 or SEQ ID NO:42～50 is operationally connected and obtained, for example, be sequence shown in SEQ IDNO:54～SEQ ID NO:56.
Another aspect of the present invention relates to a kind of recombinant vectors, its molecule that induction RNA of the present invention disturbs that is operably connected.
A kind of composition, molecule or the recombinant vectors that it contains induction RNA of the present invention interference, and pharmaceutically acceptable carrier of also relating in one aspect to of the present invention.
The method that also relates in one aspect to the molecule that obtains the induction RNA interference that suppresses epidemic encephalitis B virus of the present invention, it comprises:
(1), according to the whole genome sequence of epidemic encephalitis B virus, be chosen at the conserved sequence in gene 1 type, 2 types, 3 types and 4 type strain isolateds, as potential target sequence;
(2) the potential target sequence in (1) and human genome database are compared, the sequence of other encoding sequence or expressed sequence tag homology in eliminating and human genome, using remaining sequence as target sequence; With
(3) molecule that the synthetic induction of the target sequence RNA obtaining according to step (2) disturbs.
Wherein said conserved sequence refers in the whole genome sequence of gene 1 type, 2 types, 3 types and 4 type strain isolateds more than 80% conservative sequence, and in the whole genome sequence of gene 1 type and 3 type strain isolateds more than 90% conservative sequence.In one embodiment of the invention, be more than 95% conservative sequence in the whole genome sequence at gene 1 type and 3 type strain isolateds.
Wherein, the target sequence described in step (3) is selected from the transcripton sequence of two or more epidemic encephalitis B virus albumen.
In embodiments of the invention, the transcripton sequence of wherein said two or more epidemic encephalitis B virus albumen is selected from:
(1) membranin M and NS1 albumen;
(2) NS2A, NS2B, NS3, NS4A and NS4B albumen; Or
(3) membranin M, NS1, NS2A, NS2B, NS3, NS4A and NS4B albumen.
Molecule or the purposes of recombinant vectors in the composition for the preparation of the genetic expression of inhibition epidemic encephalitis B virus that also relates in one aspect to induction RNA of the present invention interference of the present invention.
Molecule that induction of the present invention RNA disturbs or the recombinant vectors of also relating in one aspect to of the present invention is in the purposes for the preparation for the treatment of and/or preventing in the medicine of epidemic encephalitis type B.
Of the present inventionly also relate in one aspect to molecule that induction of the present invention RNA disturbs in the purposes for the preparation of suppressing in the siRNA of epidemic encephalitis B virus.
Of the present invention also relate in one aspect to suppress epidemic encephalitis B virus siRNA for target sequence, described target sequence is expressed as at least one in following sequence with cDNA:
(1) arbitrary sequence shown in SEQ ID NO:1～SEQ ID NO:9 and SEQ ID NO:33～SEQ IDNO:50;
(2) with SEQ ID NO:1～SEQ ID NO:9, SEQ ID NO:33～SEQ IDNO:50 in the identity of arbitrary sequence be at least 70% sequence; Preferably at least 80%, more preferably at least 84%, more preferably at least 89%, more preferably at least 94%.
The siRNA molecule that also relates in one aspect to of the present invention, it contains the RNA sequence corresponding or complementary with target sequence of the present invention.
Described correspondence refers to the T in the target sequence representing with cDNA is replaced with to U; Described complementation refers to according to A-U well known in the art, G-C, C-G, T-A principle and target sequence carries out complementation, obtains RNA sequence.
In the present invention, the molecule that induction RNA disturbs is converted into the effect siRNA molecule of implementing inhibition of gene expression in cell or body.
SiRNA molecule comprises positive-sense strand and antisense strand, and being applicable to siRNA molecule of the present invention can prepare by the conventional method in this area.Can prepare in vitro siRNA, then by (for example, by transfection) in its direct transfered cell.More specifically, for example can by chemosynthesis, in-vitro transcription or by siRNA expression plasmid or virus vector at cells, prepare siRNA molecule of the present invention.
In embodiments of the invention, sequence shown in SEQ ID NO:11～SEQ ID NO:28 is connected into respectively in siRNA expression vector, carrier transfered cell, to express siRNA molecule, is disturbed to the expression of encephalitis b virus gene.
In embodiment of the present invention, by after siRNA expression vector transfered cell, siRNA expresses to be similar to the mode of endogenous miRNA, be after the source transcript of RNA is transcribed under the effect of rna plymerase ii, cut into the precursor of 60-70nt by Drosha in nucleus after, by Exportin-5, be transported in tenuigenin, in tenuigenin, siRNA precursor is cut into ripe siRNA molecule by Dicer, and ripe siRNA molecule is exercised the effect of cutting target mRNA.
In embodiments of the invention, according to inhibition, pick out inhibition best, for a kind of siRNA molecule of target sequence, for example NS1-447, adopts multiple copied, for example 3,6,9 copies are connected, further to strengthen inhibition.
In embodiments of the invention, will connect for siRNA molecule heterogeneic, part or all of target sequence, for example siRNA * 4 in the present invention, siRNA * 5 or siRNA * 9, and at cells, experiment showed, that it has the effect of better inhibition of gene expression.
Can adopt the mode of above two kinds of method combined utilization, for a plurality of genes, adopt multiple copied, to strengthen inhibition simultaneously.
In the present invention, term " is operably connected " and refers to nucleic acid and other nucleotide sequence are placed to the related state of tool in function.This can be that interconnective gene is connected by this way with control sequence, makes when suitable molecule is incorporated into control sequence, and the expression of this gene becomes feasible.For example, if the transcribing of promotor control coding sequence, this promotor will be combined with this sequence operably so.Conventionally, term " is operably connected " and refers to that the DNA sequence dna being connected is adjacent, and in the situation of secretion property leader sequence, is adjacent and in reading frame.The connection of described sequence is by connecting to implement in suitable restriction enzyme sites.If described site does not exist, can use synthetic according to conventional methods oligonucleotide adapter or joint.
In the present invention, siRNA (siRNA) refers to comprise an about 10-60 Nucleotide (or nucleotide analog), can guide or RNA (or RNA analogue) that mediate rna disturbs.SiRNA comprises double-stranded siRNA and strand siRNA, generally refers in the present invention and comprises positive-sense strand and antisense strand by double-stranded siRNA.
In the present invention, target sequence, except comprising shown in SEQ ID NO:1～SEQ ID NO:9 sequence, also comprises sequence corresponding with sequence shown in SEQ IDNO:1～SEQ ID NO:9 in the whole genome sequence of other encephalitis b virus strain that sequence is different therewith.
In the present invention, described identity refers to the shared ratio of identical Nucleotide in the corresponding window of two sequences relatively, the size of described window refers to the window that 15 above Nucleotide form, preferably at least 17, more preferably at least about 18 or 19 to 21-23 or the window of 24-29 Nucleotide, described identity at least 70%, refers to that in corresponding window at least 70% Nucleotide is identical, preferably at least 80%, more preferably at least 84%, more preferably at least 89%, more preferably at least 94%, for example 95%, 96%, 97%, 98%, 99% or 100%.Wherein different Nucleotide is preferably placed at 5 of sequence ' and/or 3 ' end, be for example positioned at apart from 5 ' and/or 1-4 nucleotide sequence of 3 ' end.
In the present invention, the genetic expression of described inhibition epidemic encephalitis B virus, is finger protein, and DNA, and/or the observable reduction of rna level or disappearance are for example reduced by least approximately 50%, 60%, 70%, 80%, 90%, 95%, 99% or more express.The result suppressing can confirm by detecting phenotype or the biochemical technology of cell or body, described biochemical technology is for example Northern hybridization, gene chip, enzyme linked immunosorbent assay (ELISA), Western Blot, fluorescence-activated cell sorting (FACS) or radioimmunoassay (RIA) etc.
The beneficial effect of the invention
In the present invention, application RNAi technology, by the siRNA for encephalitis b virus genome conserved regions, carry out the inhibition of encephalitis b virus and study, for suppressing various different genotype encephalitis b virus, copy the infection causing new approach is provided, also for the prevention of encephalitis and treatment provide new thinking.Effectively siRNA target site not only can be used as new chemicals target site, and siRNA can be directly as effectively medicine is for prevention and the treatment of encephalitis by force, and high specificity, is difficult for causing side reaction, convenient drug administration, quick.The drug combination of many siRNAs has not only further increased the effect of the Antiencephalitis vi of its wide spectrum, and provides assurance for the escape sudden change of pre-anti-virus.The siRNA medicine of the present invention's development at present and exploitation treatment encephalitis not only has actual operability but also has good application prospect.
Accompanying drawing explanation
Fig. 1 siRNAs expression vector pcDNA 6.2-GW/EmGFP-miR schematic diagram
Fig. 2 Photinus pyralis LUC siRNAs reporter plasmid carrier schematic diagram
The inhibition figure that Fig. 3 siRNAs expression vector is expressed target sequence, wherein NC contrast is the cell of the irrelevant siRNA of transfection and luciferase expression carrier, mock is the cell of transfection luciferase expression carrier untransfected siRNAs expression vector, other can be expressed for PrM for transfection, NS1, NS2A, NS2B, NS3, the cell of the special siRNA of NS4A target sites different from NS4B gene and luciferase expression carrier.Ordinate zou represents to take that the renilla luciferase of transcriptional activity internal reference is that standardized Photinus pyralis LUC is active.
Fig. 4 siRNAs expresses the series connection schematic diagram of framework
Fig. 5 flow cytometer detects the cell per-cent of the siRNA transfection positive, and wherein left figure is the cell of untransfected siRNA, right figure the has been transfection cell of siRNA expression vector NS1-447.
The special siRNAs expression vector of Fig. 6 transient transfection suppresses situation to encephalitis b virus SA14-14-2 pnca gene group RNA.Wherein NC is transfection negative control siRNA expression vector, and Mock is the contrast of untransfected siRNAs.Three of each data representations in figure repeat the mean value of experiment.
The inhibition situation map of Fig. 7 siRNA expression vector to virus envelope albumen E protein expression.Wherein, swimming lane 1-13 represents that BHK-21 cell is at transfection PrM-54, NS1-447 successively, NS1-630, NS1-1207, NS2A-461, NS2B-123, NS3-1112, NS4A-289, NS4B-115, siRNAs * 4, siRNAs * 5, the expression of siRNAs * 9 and NC contrast postoperative infection encephalitis b virus SA 14-14-2 envelope protein E albumen; The expression of envelope protein E albumen in the BHK-21 cell of swimming lane 14 expression untransfected plasmids; Swimming lane 15 is the BHK-21 cell contrast that untransfected plasmid does not infect encephalitis b virus.Actin is as internal reference.
The inhibition situation comparison of the BHK-21 cell that Fig. 8 infects latter 24 hours stably express NS1-447, (NS1-447) * 6, siRNAs * 9 and NC to encephalitis b virus SA14-14-2 pnca gene group RNA.Wherein, Mock is that common BHK-21 infects viral contrast.Three of each data representations in figure repeat the mean value of experiment.
The inhibition situation of Fig. 9 stable expression cell line to virus envelope albumen E albumen.Wherein, the inhibition situation of the clone that swimming lane 1-4 represents respectively stably express siRNAs * 9, (NS1-447) * 6, NS1-447 and NC contrast successively to virus envelope albumen E albumen, swimming lane 5 infects viral contrast for common BHK-21, and swimming lane 6 is the contrast of common BHK-21 cell uninfecting virus.Actin is internal reference.
The inhibition situation comparison of the BHK-21 cell that Figure 10 infects latter 48 hours stably express NS1-447, (NS1-447) * 6, siRNAs * 9 and NC to encephalitis b virus SA14-14-2 strain amplification titre.Mock is that common BHK-21 infects viral contrast.Three of each data representations in figure repeat the mean value of experiment.
Figure 11 infects the survival condition contrast of the BHK-21 cell of latter 96 hours stably express NS1-447, (NS1-447) * 6, siRNAs * 9 and NC.Mock is that common BHK-21 infects viral contrast.Cell control is the BHK-21 cell of uninfecting virus.Three of each data representations in figure repeat the mean value of experiment.
The inhibition situation of Figure 12 stable expression cell line to viral SA14, SH53 and SH101 envelope protein E albumen.In figure, the inhibition situation of the clone that swimming lane 1-3 represents respectively stably express siRNAs * 9, (NS1-447) * 6 and NC contrast successively to virus envelope albumen E albumen, swimming lane 4 infects viral contrast for common BHK-21, and swimming lane 5 is the contrast of common BHK-21 cell uninfecting virus.Actin is internal reference.
The inhibition situation of the BHK-21 cell that Figure 13 infects latter 48 hours stably express siRNAs * 9, (NS1-447) * 6 and NC to encephalitis b virus SA14, SH53 and SH101 titre.Mock is that common BHK-21 infects viral contrast.Three of each data representations in figure repeat the mean value of experiment.
Figure 14 stable expression cell line is in SA14, SH53 or as a child survival rate detection of SH101 virus infection 96.Infect the survival condition contrast of the BHK-21 cell of latter 96 hours stably express siRNAs * 9, (NS1-447) * 6 and NC.Mock is that common BHK-21 infects viral contrast.Cell control is the BHK-21 cell of uninfecting virus.Three of each data representations in figure repeat the mean value of experiment.
Embodiment
Below in conjunction with embodiment, embodiment of the present invention are described in detail, but it will be understood to those of skill in the art that the following example is only for the present invention is described, and should not be considered as limiting scope of the present invention.Unreceipted actual conditions person in embodiment, carries out according to the condition of normal condition or manufacturers's suggestion.The unreceipted person of production firm of agents useful for same or instrument, being can be by the conventional products of commercial acquisition.
the selection of embodiment 1siRNA target site
The whole genome sequence of analyzing all encephalitis b virus of including in GenBank amounts to 64, comprising 34 Chinese pathogenic strains.Use SeqMan software to do sequence analysis, be chosen at conservative sequence in more than 80% global strain isolated, 95% above Chinese pathogenic strain and design siRNAs target sequence.The corresponding online design software that target sequence design is used INVITROGEN to provide according to its siRNAs expression vector designs
(
http://rnaidesigner.invitrogen.com/rnaiexpress/setOption.do？des ignOption＝mirna&pid＝509133211138749536)。Designed rear use BLAST (
www.ncbi.nlm.nih.gov), potential target sequence and human genome database are compared, get rid of the sequence of those and other encoding sequences or EST homology.Design one group of negative control siRNAs (negative control, NC) simultaneously.Table 1 is for encephalitis b virus PrM, NS1, NS2A, NS2B, NS3, the target sequence of the siRNAs of NS4A and NS4B gene coding region.
Table 1 is for the target sequence of the siRNAs of encephalitis b virus gene coding region
the structure of embodiment 2siRNAs expression plasmid
In the present invention, use invitrogen company (
http:// zh.invitrogen.com/site/cn/zh/home.html) siRNAs that filters out of the pcDNA6.2-GW/EmGFP-miR vector expression that provides, according to this carrier to expressing the requirement of siRNAs sequential structure, entrust Qing Ke biotech firm (
www.tsingke.com) synthetic dsdna sequence is as shown in table 2, in table, underlined base is positive-sense strand and the antisense strand of the siRNAs corresponding with respective target sequence.Positive antisense strand equal proportion is mixed latter 95 ℃ and is hatched incubated at room annealing formation two strands after 4 minutes.The cohesive end (Fig. 1) that the ligation mediating by T4DNA ligase enzyme connects into pcDNA 6.2-GW/EmGFP-miR carrier by two strands forms a required siRNAs expression vector of use CMV promoter expression.Wherein EmGFP as the label of siRNAs expression amount and the expression level for the identification of siRNAs.
Table 2 is expressed siRNAs required synthetic DNA fragmentation positive-sense strand and antisense strand
In order to analyze the interference effect of siRNAs to each target sequence, contriver has built a series of reporter plasmids, be about to listed 9 target sequences of embodiment 1 and be cloned into respectively a Photinus pyralis LUC (Firefly Luciferase after by chemosynthesis, Fluc) expression vector is (purchased from Genordia AB, Sweden) in (as shown in Figure 2), concrete grammar is, the DNA sequence dna after annealing of the positive antisense strand of chemosynthesis target sequence forms two strands respectively, then target sequence is cloned into the Fluc gene 5 ' end of Photinus pyralis LUC, form fusion gene (as 1-Fluc, 2-Fluc), this plasmid enters cell through transfection, in cell, can express the Fluc of fusion.If target sequence is disturbed the expression amount of Fluc to reduce by siRNAs, can judge accordingly whether specificity is disturbed target sequence to siRNA.
embodiment 4siRNAs expression vector suppresses target sequence expression effect separately in BHK-21 cell
identify and screening
(1) cell transfecting
With 24 porocyte culture plates, carry out cell transfecting experiment.
1. in the day before yesterday of transfection of every hole inoculation 6 * 10 on 24 porocyte culture plates
4individual BHK-21 cell, 5%CO
237 ℃ of overnight incubation of incubator.During to transfection, cell is paved with the 80%-90% of hole area.
2. cationic-liposome Lipofectamine 2000 (Invitrogen) transfection reagent for the transfection of target sequence-Fluc integrative gene expression vector, carries out transfection experiment by its operation instructions.Concrete consumption is that every porocyte adds target sequence-Fluc integrative gene expression vector 170ng, renilla luciferase (Renilla Luciferase as transcriptional activity internal reference, RLuc) expression vector pRL-TK (Promega company product) 17ng, siRNAs expression vector pcDNA 6.2-GW/EmGFP-miR 400ng, Lipofectamine 2,000 2 μ l.
3. after 6 hours, adding foetal calf serum to final concentration is 10%.In 5%CO
2, 37 ℃ detect after continuing to be cultured to 48 hours.
(2) fluoroscopic examination
Two fluorescence report systems (Dual-luciferase Reporter AssaySystem) of application Promega company are carried out fluoroscopic examination on fluorescence illumination instrument TD-20/20 luminometer (Turnur Designs, USA).
After transfection, 48hr harvested cell carries out fluoroscopic examination.Operation instructions according to Dual-luciferase ReporterAssay System is carried out.Concrete operation step is:
1. abandon cell culture fluid, with phosphoric acid buffer (PBS), rinse cell twice, to remove cast-off cells and residual cell culture fluid.
2. in every hole, add 100 μ l 1 * lysis buffers (Passive Lysis Buffer).
3. Tissue Culture Plate is put and on oscillator, is shaken cracking 15min.
4. get the cell pyrolysis liquid 20 μ l in each cell cultures hole, add in each hole of fluorescence illumination instrument check-out console used.
5. in each sample well of check-out console, add 100 μ l Luciferse Assay Reagent II, mix and with fluorescence illumination instrument, carry out fluorescence intensity detection afterwards.
6. in each sample well, add rapidly 100 μ l Stop & Glo Reagent, then carry out fluorescence intensity detection with fluorescence illumination instrument.
7. carrying out data calculates and analyzes (according to the explanation of the Part#TM040 of Promega company technical manual, carrying out).
(3) for encephalitis b virus PrM, NS1, NS2A, NS2B, NS3, the screening method of the siRNA of NS4A and NS4B encoding gene target sequence.
By target sequence-Fluc integrative gene expression vector cotransfection BHK-21 cell of siRNAs expression vector pcDNA 6.2-GW/EmGFP-miR, the pRL-TK of embodiment 2 preparations and embodiment 3 preparations, after 48 hours, harvested cell carries out fluoroscopic examination and data calculating and analysis.Experimental result is shown in Fig. 3, table 3.
(4) result:
From the result of Fig. 3 and table 3, according to nine siRNAs of the target sequence design of screening, all the expression amount of target sequence can be suppressed at below 20%.
The inhibition that table 3siRNAs expression vector is expressed target sequence
embodiment 5siRNAs expresses the series connection of framework
As shown in Figure 4, be chosen in the best siRNAs NS1-447 of effect in embodiment 4, enzyme by BamHI, Bgl II and Xho I is cut ligation, built the siRNAs expression vector (NS1-447) * 3 (SEQ ID NO:51) that contains respectively three, six and nine NS1-447 expression frameworks, (NS1-447) * 6 (SEQ ID NO:52) and (NS1-447) * 9 (SEQ ID NO:53).Again all nine siRNAs are connected simultaneously, expression vector siRNAs * 4 (SEQ ID NO:54) of containing respectively four, five and nine siRNAs expression frameworks have been formed, siRNAs * 5 (SEQ IDNO:55), and siRNAs * 9 (SEQ ID NO:56).Above six kinds of sequence positive-sense strands of expressing framework are referring to SEQ ID NO:51～SEQ ID NO:56, wherein the sequence identical with positive-sense strand in SEQ ID NO:11～SEQ ID NO:28 is composition sequence (black matrix part in sequence table), and other sequence is carrier sequence.Concrete construction process is referring to expression vector specification sheets.
(1) cell transfecting
With 24 porocyte culture plates, carry out cell transfecting experiment.
1. in the day before yesterday of transfection of every hole inoculation 6 * 10 on 24 porocyte culture plates
4individual BHK-21 cell, 5%CO
237 ℃ of overnight incubation of incubator.During to transfection, cell is paved with the 80%-90% of hole area.
2. use cationic-liposome Lipofectamine 2000 (Invitrogen) transfection reagent, by its operation instructions, carry out transfection experiment.Concrete consumption is that every porocyte adds siRNAs expression vector pcDNA6.2-GW/EmGFP-miR 800ng, Lipofectamine 2,000 2 μ l.
3. after 6 hours, adding foetal calf serum to final concentration is 10%.In 5%CO
2, 37 ℃ continue to be cultured to next day.
(2) low cytometric analysis detects the transfection efficiency of siRNAs expression vector
The mono-expression vector of transfection siRNA, after 48 hours, is abandoned substratum, uses flow cytometer to detect the per-cent that EmGFP positive cell accounts for total cell count after peptic cell, judges successively the per-cent of siRNA transfection positive cell, and result as shown in Figure 5.
(3) virus infection
Second day after transfection completes, the substratum in cell cultures hole is abandoned in suction, use 0.1MOI (Multiplicity of infection, the ratio of virus and cell quantity when implication is infection) gene 3 type encephalitis b virus attenuated live vaccine SA14-14-2 cells infecteds were changed fresh culture after 2 hours, in 5%CO
2, 37 ℃ continue to cultivate.
(4) use fluorescent quantitative PCR technique to detect virus genome RNA and copy situation
1. after virus infection 24 hours, use the RNeasy Mini Kit (74104) of QIAGEN company to extract total RNA of every porocyte quantitatively.
2. total RNA 2 micrograms after getting quantitatively, are used Superscript III ReverseTranscriptase (invitrogen) to carry out the reverse transcription of 20 microlitre systems, the total RNA reverse transcription obtaining are become to the cDNA of 20 microlitre strands.
3. from 20 microlitre cDNA samples, respectively get 1 microlitre, use the ABIPrism 7000 Real-time PCR system systems of Applied Biosystems, carry out the fluorescent quantitation reaction of 50 microlitre systems, reaction system is as follows: 2 microlitre cDNA, upstream and downstream primer (is respectively encephalitis b virus object fragment upstream and downstream primer, i.e. upstream primer: 5 '-CCTCCGTCACCATGCCAGTCTTAG-3 ' (SEQ IDNO:31); Downstream primer: 5 '-TTCGCCATGGTCTTTTTCCTCTCG-3 ' (SEQ IDNO:32)) each 1 microlitre, 25 microlitre SYBR Green PCR Master Mix (Applied Biosystems). after completion of the reaction.Process experimental result (treatment process refers to ABI company absolute quantitation operational manual) and (in table 4, take Mock as 100%) as shown in Fig. 5, table 4.
(5) use the expression of Western blot technology for detection virus envelope albumen (Envelope, E).
1. after virus infection 24 hours, harvested cell, was used centrifuging and taking supernatant harvested cell total protein after universal cell pyrolysis liquid lysing cell.
2. get after 20 microlitre cell pyrolysis liquids mix with 5 microlitre 5 * Loading Buffer and boil 5 minutes.The sample of handling well is done protein electrophorese with 12% SDS PAGE.
3. with half-dried, transfer from one department to another to unite by the protein delivery of glue separation to NC film, use the monoclonal antibody (purchased from Hua Da protein center) of the anti-E albumen in mouse source to hatch one hour as primary antibodie, use two anti-hatching one hour of fluorescently-labeled sheep anti mouse instead.Finally use Li-Cor Odyssey system scan NC film, obtain the expression of E albumen in each sample, Actin is as the internal reference of experiment, and result as shown in Figure 6.
(6) result:
From Fig. 6, the result of table 4 and Fig. 7, the siRNA expression vector of transient transfection can suppress to a certain extent encephalitis b virus attenuated live vaccine SA14-14-2 and copy and breed intracellular, what consider that siRNA expression vector embodies in transient transfection is lower transfection efficiency (Fig. 5 shows the siRNA of the cell successful expression of only having 32.9%), contriver thinks that the screen siRNA obtaining is effectively, and the inhibition of the siRNA tandem expression carrier that wherein 9 kinds of siRNA combinations obtain is best.
Table 4siRNA expression vector suppresses situation to virus genome RNA
The clone of embodiment 7 stably express siRNA molecules builds
The negative impact bringing for eliminating the low transfection efficiency of transient transfection in embodiment 6, be chosen in best NS1-447 and six aggressiveness (NS1-447) * 6 thereof of effect in embodiment 4 and embodiment 6, and best these three carriers of siRNAs * 9 and the NC contrast of effect builds stable cell lines.Four constructed stable BHK-21 cells can be distinguished stably express NS1-447, (NS1-447) * 6, siRNAs * 9 and NC contrast (construction process refers to invitrogen operational manual K4936-00).
(1) four kinds of stable expression cell line NS1-447, (NS1-447) * 6, siRNAs * 9 and NC are inoculated in to 24 orifice plates or 96 orifice plates with identical quantity.
(2) second day, when cell approaches 90%-100% density, is used the encephalitis b virus SA 14-14-2 cells infected of 0.1MOI to change fresh culture after 2 hours, in 5%CO
2, 37 ℃ continue to cultivate.
(3) infect the latter 24 hours monolayer cells in results 24 orifice plates and according to the method for embodiment 6, detect the situation that copies of virus genome RNA, result is as shown in Fig. 8, table 5.
(4) infect the latter 36 hours monolayer cells in results 24 orifice plates and according to the method in embodiment 6, detect the expression of encephalitis b virus envelope protein E albumen, result as shown in Figure 9.
(5) infect rear 48 hour cells and start a large amount of pathologies, now gather in the crops cells and supernatant in 24 orifice plates, use Karber method to detect virus titer TCID
50.Method is as follows:
1. will after healthy cell digestion, with maintenance medium (containing the DMEM substratum of 2%FBS) dilution, become 10
5after the working concentration of/ml, by the 100 every holes of μ l, add in 96 orifice plates and mix.
2. virus stock solution used is done in sterilizing EP pipe to 10 times of continuous dilutions, use 1ml suction pipe to draw 100 μ l virus stock solution useds, add in first EP pipe that 900 μ l maintenance mediums are housed, vibration mixes, separately change a new 1ml suction pipe, after piping and druming, draw again 100 μ l and add the second pipe to be equipped with in the EP pipe of 900 μ l maintenance mediums, change suction pipe, as above fully after vibration and piping and druming evenly, then draw into the 3rd EP pipe.So operation, can make 10 times of continuous gradient dilutions continuously.
3. draw each dilution virus liquid 100 μ l, add in 96 orifice plates of completing cell, each dilution gradient is inoculated 8 holes, separately gets two holes and adds 100 μ l containing viral maintenance medium, as healthy cell, not contrast.
4. standing cultivation in 37 ℃ of carbonic acid gas incubators, day by day observed and recorded pathology situation is continuous seven days, the virus multiplication situation that can see in the 7th day, counting 50% above cell hole occurs that the dilution gradient of pathology appears in cytopathic dilution gradient and 50% following cell hole, according to Karber formula, calculates TCID
50value.Karber formula is: 1gTCID
50=L-d (s-0.5).The logarithm of the high dilution of L=wherein; The logarithmic value of d=dilution gradient, i.e. log
1010=1; The ratio summation of the positive pathology bore portion of s=.Titre result is as shown in Figure 10, table 6.
(6) infect latter 96 hours, observation of cell survival condition under inverted microscope, and in the cell of 96 orifice plates, every hole adds 20 microlitre MTS mixed solutions, detects cell proliferation situation, result is as shown in Figure 11, table 7.
(7) from the result of this experiment, after the negative impact that the low transfection efficiency of eliminating transient transfection brings, constructed stable expression cell line, the stable expression cell line of all expression NS1-447, (NS1-447) * 6 or siRNAs * 9 all can be survived under the attack of encephalitis b virus attenuated live vaccine SA14-14-2.And in these clone kinds, the titre of the geneome RNA expression amount of virus, expressing quantity and virus has all obtained great reduction.Experimental results show that the siRNA that obtains of screen to the inhibition of the attenuated live vaccine of encephalitis b virus gene 3 types and be really effectively to the protection of cell.
The inhibition situation of table 5 stable expression cell line to viral RNA
The inhibition situation of table 6 stable expression cell line to virus titer
Mock is that common BHK-21 infects viral contrast.
Table 7 stable expression cell line detects in 96 hours survival rates of virus infection
Mock is that common BHK-21 infects viral contrast.Cell control is the BHK-21 cell of uninfecting virus.
Be used in encephalitis b virus gene 3 type wild-type strain SA14 and gene 1 type wild-type strain SH53 and the SH101 of China's Mainland separation, detected stably express screen the siRNA that obtains with and series connection after the inhibition of siRNAs * 9 and (NS1-447) * 6 to these a few strain wild-type virus.
Use, with the similar detection means of embodiment 8, has detected virus envelope albumen E albumen, virus titer and cell proliferation situation, and result is as shown in Figure 12, Figure 13, table 8, Figure 14 and table 9.
Result: constructed stable expression cell line, the stable expression cell line of expressing (NS1-447) * 6 or siRNAs * 9 all can be survived under the attack of encephalitis b virus gene 3 type wild-type strain SA14 and gene 1 type wild-type strain SH53 and SH101.And in these clones, the titre of the geneome RNA expression amount of virus, expressing quantity and virus has all obtained great reduction.Experimental results show that the siRNA that obtains of screen to the inhibition of the wild-type strain of encephalitis b virus gene 3 types and gene 1 type and be really effectively to the protection of cell.
The inhibition situation of table 8 stable expression cell line to viral SA14, SH53 and SH101 titre
Mock is that common BHK-21 infects viral contrast.
Table 9 stable expression cell line detects at SA14, SH53 or 96 hours survival rates of SH101 virus infection
Mock is that common BHK-21 infects viral contrast.Cell control is the BHK-21 cell of uninfecting virus.
The siRNA that embodiment 10 screenings obtain is to encephalitis b virus gene 2 types and the inhibiting preliminary experiment of gene 4 C-type virus C
According to principle of design of the present invention, designed siRNA is not only extremely conservative in gene 1 type and gene 3 type strains, and gene 2 types and also relative conservative the sudden change of base (but can exist in target sequence) in gene 4 types, designed siRNA also has good inhibition to these two genotypic strains.At this, according to the complete sequence of the strain of gene 2 types that provide in GenBank and gene 4 types, in gene 2 types and gene 4 type strains, there is sequence after base mutation (as table 10 in the target sequence that these 9 siRNA have been synthesized in design, shown in table 11, wherein with the italic base of underscore, represent that gene 2 types compare with the target sequence designed with the present invention in gene 4 types the base that has sudden change), then be built in Photinus pyralis LUC siRNA reporter plasmid as shown in Figure 2, the plasmid building and internal reference plasmid pRL-TK and siRNAs * 9 are detected to the inhibition situation of target sequence after pair sudden change of siRNAs * 9 after by the method cotransfection of embodiment 4, inhibition is as table 10, shown in table 11, per-cent represents the expression amount of target sequence.
9 target sequences of table 10 gene 2 types and the expression amount of target sequence under siRNA effect
9 target sequences of table 11 gene 4 types and the expression amount of target sequence under siRNA effect
Result: the effect from table if the sudden change of target sequence occurs in 5 of sequence ' or 3 ' end, and is single base mutation, can't produce too large weakening to inhibition.If sudden change occurs in the centre of siRNA target sequence or the base mutation of multidigit point, inhibition can be had a greatly reduced quality and even be disappeared.But the weakening for the inhibition of a certain target sequence of same gene type can be made up by the inhibition of other target sequences.So the present invention screens siRNAs * 9th obtaining, can produce inhibiting preferably to the encephalitis b virus of gene 2 types and gene 4 types.
Simultaneously, those skilled in the art can know by inference, if 9 siRNA sequences of the present invention's design are carried out according to the gene order of gene 2 types and gene 4 type encephalitis b virus to the base mutation in corresponding site, the siRNA obtaining thus will obtain the similar inhibition with embodiment of the present invention 4-9.
The identity result of the target sequence of another attached gene 2 types and 4 types and gene 1,3 type comparisons, in Table 12.
The identity of the target sequence of table 12 gene 2 types and 4 types and gene 1,3 type comparisons
Wherein the italic with underscore is the base different from gene 1,3 types.
Although the specific embodiment of the present invention has obtained detailed description, it will be understood to those of skill in the art that.According to disclosed all instructions, can carry out various modifications and replacement to those details, these change all within protection scope of the present invention.Four corner of the present invention is provided by claims and any equivalent thereof.
Claims (10)
1. the molecule that induction RNA disturbs, it is comprised of positive-sense strand and antisense strand, it is characterized in that described positive-sense strand is the sequence shown in SEQ ID NO:2.
2. the molecule that induction RNA disturbs, its sequence is sequence shown in SEQ ID NO:52.
3. the molecule that induction RNA disturbs, its sequence is sequence shown in SEQ ID NO:54 or SEQ ID NO:56.
4. recombinant vectors, the molecule that its induction RNA of having the right described in requirement 1-3 any one of being operably connected disturbs.
5. composition, it contains molecule that the induction RNA described in claim 1-3 any one disturbs or the recombinant vectors of claim 4, and pharmaceutically acceptable carrier.
6. the molecule that the induction RNA described in claim 1-3 any one disturbs or the purposes of the recombinant vectors of claim 4 in the composition for the preparation of the genetic expression of inhibition epidemic encephalitis B virus, the genotype of described epidemic encephalitis B virus is gene 1 type or gene 3 types.
7. the molecule that the induction RNA described in claim 1-3 any one disturbs or the recombinant vectors of claim 4 be in the purposes for the preparation for the treatment of and/or preventing in the medicine of epidemic encephalitis type B, described in cause that the genotype of the epidemic encephalitis B virus of epidemic encephalitis type B is gene 1 type or gene 3 types.
8. the purposes of the molecule that the induction RNA described in claim 1-3 any one disturbs in the siRNA for the preparation of inhibition epidemic encephalitis B virus, the genotype of described epidemic encephalitis B virus is gene 1 type or gene 3 types.
9. suppress epidemic encephalitis B virus siRNA for target sequence, described target sequence is expressed as sequence shown in SEQ ID NO:2 with cDNA.
10.siRNA molecule, it is the RNA sequence with target complement sequence claimed in claim 9.
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| WO2007044468A2 (en) * | 2005-10-05 | 2007-04-19 | The Cbr Institute For Biomedical Research, Inc. | Method to treat flavivirus infection with sirna |
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