CN102154290A - SiRNAs for inhibiting epidemic encephalitis B viruses - Google Patents
SiRNAs for inhibiting epidemic encephalitis B viruses Download PDFInfo
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- CN102154290A CN102154290A CN 201110005104 CN201110005104A CN102154290A CN 102154290 A CN102154290 A CN 102154290A CN 201110005104 CN201110005104 CN 201110005104 CN 201110005104 A CN201110005104 A CN 201110005104A CN 102154290 A CN102154290 A CN 102154290A
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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 induce RNA interferential siRNA (siRNA), 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 that causes in the world people's encephalitis.1871, Japanese reported first should disease popular, nineteen thirty-five Japan scholar is separated to this virus from the encephalitis patient's that dies of illness cerebral tissue, thus in the world with this pathogenic agent called after japanese encephalitis virus (Japanese encephalitisvirus, JEV).According to the WHO statistics, annual average morbidity 50000 examples in the whole world, dead 15000 examples.About 1/3rd patient can be therefore and dead, has the patient of neural system sequela to account for 40%-70% among the patient of survival, and this disease mainly is popular in Asia, about more than 20 countries of south east asia.Since the nineties in 20th century, the epidemic regions of encephalitis constantly enlarges, nineteen ninety-five Papua New Guinea and Australian northern island the eruption and prevalence of encephalitis appears, 1998 in Australian native country the encephalitis case appear.Encephalitis in 2005 is in India and Nepal's eruption and prevalence, and thousands of people are ill.China region is wide, accounts for 1/4th zones, Asia, and the encephalitis morbidity number of China accounts for the whole world more than 80% of number of always falling ill, and is the popular big country of encephalitis.According to " People's Republic of China's law on the prevention and control of infectious diseases ", epidemic encephalitis type B belongs to Category B notifiable disease.From 1938 by serology experiment confirm encephalitis in China popular and in 1940 after Beijing is separated to the first strain encephalitis b virus, encephalitis repeatedly breaks out within the border in China.Over past ten years,, also be not very popular annual 20000-30,000 the case load that still has though the morbidity number of encephalitis is the trend that reduces year by year.
Encephalitis B also is a kind of natural epidemic disease source sexually transmitted disease of infecting both domestic animals and human simultaneously, and pig is this sick main harm object in animal.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 about 50%.Therefore the main financial loss that should disease infected pigs causes is that breeding difficulty, the healthy and stable development of serious harm pig industry take place farrowing sow.
Still do not have the special treatment measure at encephalitis B, vaccination still is the most effective preventive measures of protection Susceptible population.Though vaccination can be protected not infection population effectively, for the encephalitis patient who has infected, lack strong treatment means, so the methods of treatment method of using 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, its genome are sub-thread positive chain RNA molecule, are about 11kb, and molecular weight is about 4 * 10
6Dalton, settling ratio is 42s.Whole genome by 5 ' end non-translational region (5 '-untranslated regions, UTR), single open reading frame of almost crossing over whole genome (open reading frame, ORF) and 3 ' end non-translational region (3 '-UTR) constitute.95 Nucleotide of 5 ' end and 586 Nucleotide of 3 ' end are non-coding region, are three structural protein from 5 ' terminal gene order to 3 ' terminal coding, comprise capsid protein C (capsid), membranin M (membrane, PrM is a precursor) and envelope protein E (envelope), seven non-structural protein NS 1s, NS2A, NS2B, NS3, NS4A, NS4B and NS5, about 3430 amino acid of encoding altogether, its genome structure is seen 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 mainly are 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 mainly are distributed in countries such as Malaysia, Indonesia and northern Australia, are endemic genotype.
RNA disturbs (RNA interfering, RNAi) technology be new development in recent years get up a kind of efficiently, the gene disruption technology of high specificity, be a kind of by double-stranded RNA (dsRNA) or microRNA (microRNA, miRNA) mediation, transcribe back mRNA level and close the sequence-specific gene silencing mechanism that corresponding gene is expressed.It also is a kind of protection mechanism that the vivo gene group is resisted external infection and inner swivel base, extensively is 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 by Dicer cut into 21-25nt siRNA s (small interfering RNAs, siRNAs).These siRNAs are in conjunction with homologous target mRNAs on RNA dependency silencing complex (RISC) the back specificity degraded sequence.For endogenous miRNAs, source transcript (the primary RNAtranscripts of RNA, pri-miRNAs) in nucleus, cut into miRNA precursor (the miRNA precursors of 60-70nt by Drosha, pre-miRNAs) back is transported in the tenuigenin by Exportin-5, in tenuigenin, pre-miRNAs is cut into sophisticated miRNAs by Dicer.Sophisticated miRNAs exercises cutting target mRNA or suppresses the effect of translation.Most pri-miRNA transcript all is arranged under the rna plymerase ii promotor with the form of cluster, with a plurality of different miRNAs of polycistronic formal representation.In mammalian cell, (short hairpin RNA, shRNA) expression vector all can be induced RNAi for the siRNAs of the 21-29nt of external source importing chemosynthesis or short hairpin RNA.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 the RNAi technology, select different target sites to use siRNA and suppress virus replication and infect open in multiple virus.At encephalitis b virus, the existing report of research in the past can effectively suppress siRNAs or the shRNAs that encephalitis b virus duplicates.But the siRNAs of these reports or shRNAs are all only at a certain strain encephalitis b virus in gene 3 types, this characteristic makes its effect be subjected to great restriction, because, because the polytropy of encephalitis b virus geneome RNA sequence, be difficult to guarantee in other strain in gene 3 types effectively at the effective siRNA of a certain strain virus in gene three types, promptly enable in gene 3 types, to be suitable for, be difficult to also guarantee that it can be suitable in other three kinds of genotype.In addition, the virus that is in for a long time under the RNAi inhibition can the sudden change of escaping occur in the position of its target sequence, and single or two siRNAs are easy to and can lose effect owing to the generation of escape sudden change.So the means of the most effective inhibition virus replication are the drug combinations at many siRNAs of encephalitis b virus conserved regions target sequence.And this scheme was not relating in the design at the siRNAs of encephalitis b virus in the past.
Summary of the invention
The present invention uses the RNAi technology, carries out the inhibition research of encephalitis b virus by the siRNA at encephalitis b virus genome conserved regions, and has verified the simultaneously applied validity of a plurality of siRNA.Particularly, the present invention includes the following aspects:
One aspect of the present invention relates to induces RNA interferential molecule, 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 the 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) the sequence in (1) or (2) of being selected from through transforming, it is to be added with the sequence of 60 Nucleotide at the most on 5 ' direction of described sequence and 3 ' direction, 5 ' direction or 3 ' direction;
(4) will be selected from the sequence that the sequence of (1)-(3) is operationally connected and obtained according to 5 '-3 ' direction, wherein operationally placed in-line sequence is a kind, and its copy number is more than 2 or 2; With
(5) will be selected from the sequence that the sequence of (1)-(3) is operationally connected and obtained according to 5 '-3 ' direction, wherein operationally placed in-line sequence is more than 2 kinds, and per a kind copy number 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%.
Wherein the Nucleotide number of 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 the sequence of (4) described sequence for 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 is sequence shown in SEQID NO:51～SEQ ID NO:53; Wherein the sequence of (5) described sequence for 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 is sequence shown in SEQ IDNO:54～SEQ ID NO:56.
Another aspect of the present invention relates to a kind of recombinant vectors, its RNA of inducing interferential molecule of the present invention that has been operably connected.
Of the present inventionly also relate in one aspect to a kind of composition, it contains the RNA of inducing interferential molecule of the present invention or recombinant vectors, and pharmaceutically acceptable carrier.
The method of inducing RNA interferential molecule that obtains to suppress epidemic encephalitis B virus that also relates in one aspect to of the present invention, it comprises:
(1), is chosen at the conserved sequence in gene 1 type, 2 types, 3 types and the 4 type strain isolateds, as the potential target sequence according to the whole genome sequence of epidemic encephalitis B virus;
(2) potential target sequence in (1) and human genome database are compared, get rid of and human genome in other encoding sequence or expressed sequence tag homologous sequence, with remaining sequence as target sequence; With
(3) the synthetic RNA interferential molecule of inducing of target sequence that obtains according to step (2).
Wherein said conserved sequence is meant the sequence of guarding more than 80% in the whole genome sequence of gene 1 type, 2 types, 3 types and 4 type strain isolateds, and the sequence of guarding more than 90% in the whole genome sequence of gene 1 type and 3 type strain isolateds.In one embodiment of the invention, the sequence in the whole genome sequence of gene 1 type and 3 type strain isolateds, guarding more than 95%.
Wherein, the target sequence described in the step (3) is selected from the proteic transcripton sequence of two or more epidemic encephalitis B virus.
In embodiments of the invention, the proteic transcripton sequence of wherein said two or more epidemic encephalitis B virus 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.
Of the present inventionly also relate in one aspect to the RNA of inducing interferential molecule of the present invention or recombinant vectors are used for suppressing the composition of epidemic encephalitis B virus genetic expression in preparation purposes.
The RNA of inducing interferential molecule of the present invention or the recombinant vectors of also relating in one aspect to of the present invention is used for the treatment of and/or prevents purposes in the medicine of epidemic encephalitis type B in preparation.
Of the present inventionly also relate in one aspect to the RNA of inducing interferential molecule of the present invention is used for suppressing the siRNA of epidemic encephalitis B virus in preparation purposes.
Of the present invention also relate in one aspect to the siRNA that suppresses epidemic encephalitis B virus at target sequence, described target sequence is expressed as at least a in the following sequence with cDNA:
(1) arbitrary sequence shown in SEQ ID NO:1～SEQ ID NO:9 and the 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 corresponding with target sequence of the present invention or complementary RNA sequence.
Described correspondence is meant that the T in the target sequence that will represent with cDNA replaces with U; Described complementation is meant according to A-U well known in the art, G-C, C-G, T-A principle and target sequence carries out complementation, obtains the RNA sequence.
In the present invention, induce RNA interferential molecule in cell or body, to be converted into the effect siRNA molecule of implementing inhibition of gene expression.
The siRNA molecule comprises positive-sense strand and antisense strand, is applicable to that siRNA molecule of the present invention can be by this area method preparation commonly used.Can be at external preparation siRNA, then with (for example by transfection) in its direct transfered cell.More specifically, for example can in cell, express, prepare siRNA molecule of the present invention by chemosynthesis, in-vitro transcription or by siRNA expression plasmid or virus vector.
In embodiments of the invention, sequence shown in SEQ ID NO:11～SEQ ID NO:28 is connected into respectively in the siRNA expression vector, the carrier transfered cell to express the siRNA molecule, is disturbed the encephalitis b virus expression of gene.
In embodiment of the present invention, behind siRNA expression vector transfered cell, siRNA expresses in the mode that is similar to endogenous miRNA, the source transcript that is RNA is after transcribing under the effect of rna plymerase ii, be transported in the tenuigenin by Exportin-5 after in nucleus, being cut into the precursor of 60-70nt by Drosha, in tenuigenin, the siRNA precursor is cut into sophisticated siRNA molecule by Dicer, and sophisticated siRNA molecule is exercised the effect of cutting target mRNA.
In embodiments of the invention, according to suppressing effect, pick out suppress effect best, at a kind of siRNA molecule of target sequence, for example NS1-447 adopts multiple copied, for example 3,6,9 copies are connected, and suppress effect with further enhancing.
In embodiments of the invention, will connect at siRNA molecule heterogeneic, part or all of target sequence, for example the siRNA among the present invention * 4, siRNA * 5 or siRNA * 9, and in cell, express, experiment showed, that it has the effect of better inhibition of gene expression.
Can adopt mode, at a plurality of genes, adopt multiple copied simultaneously, suppress effect to strengthen with above two kinds of method combined utilization.
In the present invention, term " is operably connected " and is meant nucleic acid and other nucleotide sequence are placed at the state that has relation on the function.This can be that interconnective gene is connected by this way with control sequence, makes that this expression of gene becomes feasible when suitable molecule is incorporated into control sequence.For example, if promotor is controlled transcribing of encoding sequence, this promotor combines operability ground with this sequence so.Usually, term " is operably connected " and is meant that connected dna sequence dna is adjacent, and in the situation of secretion property leader sequence, is adjacent and in reading frame.The connection of described sequence is to implement by connecting on suitable restriction enzyme sites.If described site does not exist, can use synthetic oligonucleotide adapter or joint according to conventional methods.
In the present invention, siRNA (siRNA) refers to comprise about 10-60 Nucleotide (or nucleotide analog), can guide or mediate rna interferential RNA (or RNA analogue).SiRNA comprises double-stranded siRNA and strand siRNA, generally is meant in the present invention to comprise positive-sense strand and antisense strand by double-stranded siRNA.
In the present invention, target sequence is except that comprising shown in SEQ ID NO:1～SEQ ID NO:9 the sequence, also comprises in the whole genome sequence of other encephalitis b virus strain that sequence is different therewith and the corresponding sequence of sequence shown in SEQ IDNO:1～SEQ ID NO:9.
In the present invention, described identity is meant the shared ratio of identical Nucleotide in the corresponding window of two sequences relatively, the size of described window is meant the window that 15 above Nucleotide are formed, 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% is meant 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, for example be 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 observable reduction of rna level or disappearance for example are reduced by at least about 50%, 60%, 70%, 80%, 90%, 95%, 99% or more the expression.The result who suppresses can confirm by phenotype or the biochemical technology that detects cell or body, described biochemical technology for example is 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, use the RNAi technology, carry out the inhibition research of encephalitis b virus by the siRNA at encephalitis b virus genome conserved regions, duplicate the infection that causes new approach is provided for suppressing various different genotype encephalitis b virus, also the prevention for encephalitis provides new thinking with treatment.Effectively the siRNA target site not only can be used as new chemicals target site, and siRNA can directly be used for the prevention and the treatment of encephalitis as strong effectively medicine, and high specificity is difficult for causing side reaction, convenient drug administration, fast.The drug combination of many siRNAs has not only further increased the effect of the anti-encephalitis b virus 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 prospects for application.
Description of drawings
Fig. 1 siRNAs expression vector pcDNA 6.2-GW/EmGFP-miR synoptic diagram
Fig. 2 Photinus pyralis LUC siRNAs reporter plasmid carrier synoptic diagram
The inhibition design sketch that Fig. 3 siRNAs expression vector is expressed target sequence, wherein the NC contrast is the cell of 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 at PrM for transfection, NS1, NS2A, NS2B, NS3, the cell of special siRNA of the different target sites of NS4A and luciferase expression carrier with the NS4B gene.Ordinate zou represents that the renilla luciferase with the transcriptional activity internal reference is standardized Photinus pyralis LUC activity.
Fig. 4 siRNAs expresses the series connection synoptic diagram of framework
Fig. 5 flow cytometer detects siRNA transfection positive cells per-cent, and wherein left side figure is the cell of untransfected siRNA, right side 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 a transfection negative control siRNA expression vector, and Mock is the contrast of untransfected siRNAs.The mean value of three repeated experiments of each data representation among the figure.
Fig. 7 siRNA expression vector is to the inhibition situation map of virus envelope albumen E protein expression.Wherein, swimming lane 1-13 represents that successively the BHK-21 cell is at transfection PrM-54, NS1-447, NS1-630, NS1-1207, NS2A-461, NS2B-123, NS3-1112, NS4A-289, NS4B-115, siRNAs * 4, siRNAs * 5, siRNAs * 9 and the proteic expression of NC contrast postoperative infection encephalitis b virus SA 14-14-2 envelope protein E; The proteic expression of envelope protein E in the BHK-21 cell of swimming lane 14 expression untransfected plasmids; Swimming lane 15 does not infect the BHK-21 cell contrast of encephalitis b virus for the untransfected plasmid.Actin is as internal reference.
Fig. 8 infects the inhibition situation comparison of the BHK-21 cell of back 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 the contrast of common BHK-21 infective virus.The mean value of three repeated experiments of each data representation among the figure.
Fig. 9 stable expression cell line is to the proteic inhibition situation of virus envelope albumen E.Wherein, swimming lane 1-4 represents the clone of stably express siRNAs * 9, (NS1-447) * 6, NS1-447 and NC contrast to the proteic inhibition situation of virus envelope albumen E respectively successively, swimming lane 5 is the contrast of common BHK-21 infective virus, and swimming lane 6 is the contrast of common BHK-21 cell uninfecting virus.Actin is an internal reference.
Figure 10 infects the inhibition situation comparison of the BHK-21 cell of back 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 the contrast of common BHK-21 infective virus.The mean value of three repeated experiments of each data representation among the figure.
Figure 11 infects the survival condition contrast of the BHK-21 cell of back 96 hours stably express NS1-447, (NS1-447) * 6, siRNAs * 9 and NC.Mock is the contrast of common BHK-21 infective virus.Cell control is the BHK-21 cell of uninfecting virus.The mean value of three repeated experiments of each data representation among the figure.
Figure 12 stable expression cell line is to viral SA14, SH53 and the proteic inhibition situation of SH101 envelope protein E.Among the figure, swimming lane 1-3 represents the clone of stably express siRNAs * 9, (NS1-447) * 6 and NC contrast to the proteic inhibition situation of virus envelope albumen E respectively successively, swimming lane 4 is the contrast of common BHK-21 infective virus, and swimming lane 5 is the contrast of common BHK-21 cell uninfecting virus.Actin is an internal reference.
Figure 13 infects the inhibition situation of the BHK-21 cell of back 48 hours stably express siRNAs * 9, (NS1-447) * 6 and NC to encephalitis b virus SA14, SH53 and SH101 titre.Mock is the contrast of common BHK-21 infective virus.The mean value of three repeated experiments of each data representation among the figure.
Figure 14 stable expression cell line is in SA14, SH53 or SH101 virus infection 96 survival rate detection as a child.Infect the survival condition contrast of the BHK-21 cell of back 96 hours stably express siRNAs * 9, (NS1-447) * 6 and NC.Mock is the contrast of common BHK-21 infective virus.Cell control is the BHK-21 cell of uninfecting virus.The mean value of three repeated experiments of each data representation among the figure.
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 only is used to illustrate the present invention, and should not be considered as limiting scope of the present invention.Unreceipted actual conditions person among the 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 among the GenBank amounts to 64, comprising 34 Chinese pathogenic strains.Use SeqMan software to do the homology comparison, be chosen at sequence conservative in global strain isolated more than 80%, the 95% above Chinese pathogenic strain and design siRNAs target sequence.The target sequence design uses INVITROGEN to design according to the corresponding online design software that its siRNAs expression vector provides
(
http://rnaidesigner.invitrogen.com/rnaiexpress/setOption.do？des ignOption＝mirna&pid＝509133211138749536)。Use BLAST after design is finished (
Www.ncbi.nlm.nih.gov), potential target sequence and human genome database are compared, get rid of those and other encoding sequences or EST homologous sequence.Design simultaneously one group of negative control siRNAs (negative control, NC).Table 1 is at encephalitis b virus PrM, NS1, NS2A, NS2B, NS3, the target sequence of the siRNAs of NS4A and NS4B gene coding region.
Table 1 is at 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 provided, according to this carrier to express the requirement of siRNAs sequential structure, entrust hold up biotech firm of section (
Www.tsingke.com) the synthetic dsdna sequence is as shown in table 2, underlined base is positive-sense strand and the antisense strand of the siRNAs corresponding with the respective target sequence in the table.Positive antisense strand equal proportion is mixed back 95 ℃ and is hatched incubated at room annealing formation two strands after 4 minutes.Ligation by the mediation of T4DNA ligase enzyme constitutes a required siRNAs expression vector of use CMV promoter expression with the cohesive end (Fig. 1) that two strands connects into pcDNA 6.2-GW/EmGFP-miR carrier.Wherein EmGFP is used to identify expression amount and the expression level of siRNAs the label 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, the contriver has made up a series of reporter plasmids, be about to embodiment 1 listed 9 target sequences and be cloned into a Photinus pyralis LUC (Firefly Luciferase respectively after by chemosynthesis, Fluc) expression vector is (available 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, constitute fusion gene (as 1-Fluc, 2-Fluc), this plasmid enters cell through transfection, can express the Fluc of fusion in cell.The expression amount of Fluc reduces if target sequence is disturbed then by siRNAs, can judge in view of the above whether specificity is disturbed target sequence to siRNA.
Embodiment 4siRNAs expression vector suppresses target sequence expression effect separately in the BHK-21 cell
Identify and screening
(1) cell transfecting
Carry out the cell transfecting experiment with 24 porocyte culture plates.
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.Cell is paved with the 80%-90% of hole area during to transfection.
2. the transfection of target sequence-Fluc integrative gene expression vector is carried out transfection experiment with cationic-liposome Lipofectamine 2000 (Invitrogen) transfection reagent by its operation instructions.Concrete consumption is that every porocyte adds target sequence-Fluc integrative gene expression vector 170ng, renilla luciferase (Renilla Luciferase as the 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. adding foetal calf serum to final concentration after 6 hours is 10%.In 5%CO
2, 37 ℃ detect after continuing to be cultured to 48 hours.
(2) fluoroscopic examination
Use two fluorescence report systems (Dual-luciferase Reporter AssaySystem) of Promega company, (Turnur Designs carries out fluoroscopic examination on USA) at fluorescence illumination instrument TD-20/20 luminometer.
The 48hr harvested cell carries out fluoroscopic examination after the transfection.Operation instructions according to Dual-luciferase ReporterAssay System is carried out.The concrete operations step is:
1. abandon cell culture fluid, with phosphoric acid buffer (PBS) flushing 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 concussion cracking 15min on the oscillator.
4. get the cell pyrolysis liquid 20 μ l in each cell cultures hole, add in each hole of the used check-out console of fluorescence illumination instrument.
5. add 100 μ l Luciferse Assay Reagent II in each sample well of check-out console, carry out fluorescence intensity with fluorescence illumination instrument behind the mixing and detect.
6. add 100 μ l Stop in each sample well rapidly; Glo Reagent carries out fluorescence intensity with fluorescence illumination instrument again and detects.
7. carry out data computation and analysis (carrying out) according to the explanation of the Part#TM040 of Promega company technical manual.
(3) at encephalitis b virus PrM, NS1, NS2A, NS2B, NS3, the screening method of the siRNA of NS4A and NS4B encoding gene target sequence.
With siRNAs expression vector pcDNA 6.2-GW/EmGFP-miR, the pRL-TK of embodiment 2 preparations and the target sequence-Fluc integrative gene expression vector cotransfection BHK-21 cell of embodiment 3 preparations, harvested cell carries out fluoroscopic examination and data computation and analysis after 48 hours.Experimental result is seen Fig. 3, table 3.
(4) result:
From the result of Fig. 3 and table 3 as seen, nine siRNAs according to the target sequence design of screening all can be suppressed at the expression amount of target sequence below 20%.
The inhibition effect 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 among the embodiment 4, enzyme by BamHI, Bgl II and Xho I is cut ligation, made up the siRNAs expression vector (NS1-447) * 3 (SEQ ID NO:51) that contains three, six and nine NS1-447 expression frameworks respectively, (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 four, five and nine siRNAs expression frameworks have respectively been constituted, siRNAs * 5 (SEQ IDNO:55), and siRNAs * 9 (SEQ ID NO:56).More than six kinds of sequence positive-sense strands of expressing frameworks referring to SEQ ID NO:51～SEQ ID NO:56, wherein identical with positive-sense strand among SEQ ID NO:11～SEQ ID NO:28 sequence is composition sequence (a black matrix part in the sequence table), and other sequence is the carrier sequence.Concrete construction process is referring to the expression vector specification sheets.
(1) cell transfecting
Carry out the cell transfecting experiment with 24 porocyte culture plates.
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.Cell is paved with the 80%-90% of hole area during to transfection.
2. use cationic-liposome Lipofectamine 2000 (Invitrogen) transfection reagent, carry out transfection experiment by its operation instructions.Concrete consumption is that every porocyte adds siRNAs expression vector pcDNA6.2-GW/EmGFP-miR 800ng, Lipofectamine 2,000 2 μ l.
3. adding foetal calf serum to final concentration after 6 hours 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 single expression vector of transfection siRNA was abandoned substratum after 48 hours, used flow cytometer to detect the per-cent that the EmGFP positive cell accounts for total cell count behind the peptic cell, judged the per-cent of siRNA transfection positive cell successively, and the result as shown in Figure 5.
(3) virus infection
After transfection is finished second day, the substratum in the 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 duplicate situation
1. behind the virus infection 24 hours, use the RNeasy Mini Kit (74104) of QIAGEN company to extract total RNA of every porocyte and quantitatively.
2. total RNA 2 micrograms after getting quantitatively use Superscript III ReverseTranscriptase (invitrogen) to carry out the reverse transcription of 20 microlitre systems, the total RNA reverse transcription that obtains are become 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, the upstream and downstream primer (be respectively encephalitis b virus purpose 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 reaction finishes.Handle experimental result (treatment process sees ABI company absolute quantitation operational manual for details) (in table 4 be 100% with Mock) shown in Fig. 5, table 4.
(5) use Western blot technology for detection virus envelope albumen (Envelope, expression E).
1. behind the virus infection 24 hours, harvested cell used centrifuging and taking supernatant harvested cell total protein behind the universal cell pyrolysis liquid lysing cell.
2. boiled 5 minutes after getting 20 microlitre cell pyrolysis liquids and 5 microlitres, 5 * Loading Buffer mixing.The sample of handling well is done protein electrophorese with 12% SDS PAGE.
3. with the half-dried system that transfers from one department to another the isolating albumen of glue is transferred on the NC film, uses the proteic monoclonal antibody of the anti-E in mouse source (available from magnificent larger protein center), use two anti-hatching one hour of fluorescently-labeled sheep anti mouse instead as anti-hatching one hour.Use Li-Cor Odyssey system scan NC film at last, obtain the proteic expression of E in each sample, Actin is as the internal reference of experiment, and the 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 encephalitis b virus attenuated live vaccine SA14-14-2 to a certain extent and duplicate and breed intracellular, what consider that the siRNA expression vector embodies in transient transfection is lower transfection efficiency (Fig. 5 shows the siRNA that has only 32.9% cell successful expression), the contriver thinks that the siRNA that obtains that screens is effectively, and wherein the inhibition effect of the siRNA tandem expression carrier that obtains of 9 kinds of siRNA combinations is best.
Table 4siRNA expression vector suppresses situation to virus genome RNA
The clone of embodiment 7 stably express siRNA molecules makes up
The negative impact that brings for the low transfection efficiency of eliminating transient transfection among the embodiment 6, be chosen in best NS1-447 and six aggressiveness (NS1-447) * 6 and best these three carriers of siRNAs * 9 and the NC contrast structure stable cell lines of effect of effect among embodiment 4 and the embodiment 6.Four constructed stable BHK-21 cells can be distinguished stably express NS1-447, (NS1-447) * 6, siRNAs * 9 and NC contrast (construction process sees invitrogen operational manual K4936-00 for details).
(1) four kinds of stable expression cell line NS1-447, (NS1-447) * 6, siRNAs * 9 and NC are inoculated in 24 orifice plates or 96 orifice plates with identical quantity.
(2) second days,, use the encephalitis b virus SA 14-14-2 cells infected of 0.1MOI to change fresh culture after 2 hours, in 5%CO when cell the time near 90%-100% density
2, 37 ℃ continue to cultivate.
(3) monolayer cell in back 24 hours results of infection 24 orifice plates is according to the situation of duplicating of the method detection virus genome RNA of embodiment 6, and the result is shown in Fig. 8, table 5.
(4) infect the back 36 hours monolayer cells in results 24 orifice plates and detect the proteic expression of encephalitis b virus envelope protein E according to the method among the embodiment 6, the result as shown in Figure 9.
(5) infect back 48 hour cells and begin a large amount of pathologies, gather in the crops cells and supernatant in 24 orifice plates this moment, use the Karber method to detect virus titer TCID
50Method is as follows:
1. healthy cell digestion back is become 10 with keeping liquid (the DMEM substratum that contains 2%FBS) dilution
5Behind the working concentration of/ml, add mixing in 96 orifice plates by the every hole of 100 μ l.
2. virus stock solution used is done 10 times of dilutions of successive in sterilization EP pipe, promptly use the 1ml suction pipe to draw 100 μ l virus stock solution useds, add and to be equipped with in first EP pipe that 900 μ l keep liquid, the vibration mixing, other changes a new 1ml suction pipe, draws 100 μ l after the piping and druming again and adds second pipe and be equipped with in the EP pipe that 900 μ l keep liquid, changes suction pipe, after as above abundant vibration and the piping and druming evenly, draw into the 3rd EP pipe again.So operation can be made 10 times of gradient dilutions of successive continuously.
3. draw each dilution viral liquid 100 μ l, add and to have completed in 96 orifice plates of cell, each dilution gradient is inoculated 8 holes, and other gets two holes and adds the liquid of keeping that 100 μ l do not contain virus and contrast as healthy cell.
4. in 37 ℃ of carbonic acid gas incubators, leave standstill cultivation, day by day observed and recorded pathology situation is continuous seven days, the virus multiplication situation that can see in the 7th day, the dilution gradient that pathology appears in cytopathic dilution gradient and 50% following cell hole appears in counting 50% above cell hole, according to Karber formula calculating TCID
50Value.The 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 shown in Figure 10, table 6.
(6) infected back 96 hours, observation of cell survival condition under the inverted microscope, and every hole adds 20 microlitre MTS mixed solutions in the cell of 96 orifice plates, detects the cell proliferation situation, the result is shown in Figure 11, table 7.
(7) from this result of experiment, get rid of after the negative impact that low transfection efficiency brought of transient transfection, 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 to screen the siRNA that obtains be really effectively to the inhibition of the attenuated live vaccine of encephalitis b virus gene 3 types and the protection of pair cell.
Table 5 stable expression cell line is to the inhibition situation of viral RNA
Table 6 stable expression cell line is to the inhibition situation of virus titer
Mock is the contrast of common BHK-21 infective virus.
Table 7 stable expression cell line detects in 96 hours survival rates of virus infection
Mock is the contrast of common BHK-21 infective virus.Cell control is the BHK-21 cell of uninfecting virus.
Be used in isolating encephalitis b virus gene 3 type wild-type strain SA14 in China's Mainland and gene 1 type wild-type strain SH53 and SH101, detected stably express screen the siRNA that obtains with and series connection after siRNAs * 9 and (NS1-447) * 6 to the inhibition effect of these a few strain wild-type virus.
Use has detected virus envelope albumen E albumen, virus titer and cell proliferation situation with embodiment 8 similar detection meanss, and the result is shown in Figure 12, Figure 13, table 8, Figure 14 and table 9.
The 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 to screen the siRNA that obtains be really effectively to the inhibition of the wild-type strain of encephalitis b virus gene 3 types and gene 1 type and the protection of pair cell.
Table 8 stable expression cell line is to the inhibition situation of viral SA14, SH53 and SH101 titre
Mock is the contrast of common BHK-21 infective virus.
Table 9 stable expression cell line detects at SA14, SH53 or 96 hours survival rates of SH101 virus infection
Mock is the contrast of common BHK-21 infective virus.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 Cs
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 in gene 2 types and gene 4 types also relative conservative the sudden change of base (but can exist in the target sequence), designed siRNA also has these two genotypic strains and suppresses effect preferably.At this, complete sequence according to the strain of gene 2 types that provided among the GenBank and gene 4 types, sequence behind the base mutation takes place (as table 10 in the target sequence that these 9 siRNA have been synthesized in design in gene 2 types and gene 4 type strains, shown in the table 11, wherein represent in gene 2 types and gene 4 types with the designed target sequence of the present invention and compare the base that sudden change is arranged) with the italic base of underscore, then it is built in the Photinus pyralis LUC siRNA reporter plasmid as shown in Figure 2, the inhibition situation of target sequence after pair sudden change of siRNAs * 9 will be detected behind the plasmid that build and confidential reference items plasmid pRL-TK and the siRNAs * 9 method cotransfection by embodiment 4, suppress effect such as table 10, shown in the table 11, per-cent is represented the expression amount of target sequence.
9 target sequences of table 10 gene 2 types and the expression amount of target sequence under the siRNA effect
9 target sequences of table 11 gene 4 types and the expression amount of target sequence under the siRNA effect
The result: the effect from table, the sudden change of target sequence be if occur in 5 of sequence ' or 3 ' end, and be single base mutation, can't produce too big weakening to suppressing effect.If sudden change occurs in the centre of siRNA target sequence or the base mutation of multidigit point, suppress effect and can have a greatly reduced quality even disappear.But the weakening at the inhibition effect of a certain target sequence of same genotype can be remedied by the inhibition of other target sequences.So the present invention screens siRNAs * 9th that obtains, can produce good inhibitory effect to the encephalitis b virus of gene 2 types and gene 4 types.
Simultaneously, those skilled in the art can know by inference, if according to the gene order of gene 2 types and gene 4 type encephalitis b virus 9 siRNA sequences of the present invention's design are carried out the base mutation in corresponding site, the siRNA that obtains thus will obtain similarly to suppress effect with embodiment of the invention 4-9.
The target sequence of attached gene 2 types and 4 types and gene 1,3 types identity result relatively sees Table 12 in addition.
The target sequence of table 12 gene 2 types and 4 types and gene 1,3 types identity relatively
Wherein the italic with underscore is and the different base of 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 (15)
1. induce RNA interferential molecule, 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 the 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) the sequence in (1) or (2) of being selected from through transforming, it is to be added with the sequence of 60 Nucleotide at the most on 5 ' direction of described sequence and 3 ' direction, 5 ' direction or 3 ' direction;
(4) will be selected from the sequence that the sequence of (1)-(3) is operationally connected and obtained according to 5 '-3 ' direction, wherein operationally placed in-line sequence is a kind, and its copy number is more than 2 or 2; With
(5) will be selected from the sequence that the sequence of (1)-(3) is operationally connected and obtained according to 5 '-3 ' direction, wherein operationally placed in-line sequence is more than 2 kinds, and per a kind copy number is at least 1.
Claim 1 induce RNA interferential molecule, wherein the identity of (2) preferably at least 80%, more preferably at least 84%, more preferably at least 89%, more preferably at least 94%.
Claim 1 induce RNA interferential molecule, wherein the Nucleotide number of adding in (3) is 50 at the most, preferably at the most 40, more preferably at the most 30, more preferably at the most 20, most preferably at the most 10.
4. claim 1 induces RNA interferential molecule, wherein the sequence of (4) described sequence for the sequence of SEQ ID NO:2, the SEQ ID NO:34 of 3,6 or 9 copies or SEQ ID NO:43 is operationally connected and obtained for example is sequence shown in SEQ ID NO:51～SEQ ID NO:53; Wherein the sequence of (5) described sequence for the sequence of SEQ ID NO:1～4, SEQ ID NO:5～9, SEQ ID NO:1～9, SEQ ID NO:33～36, SEQ ID NO: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 is sequence shown in SEQ ID NO:54～SEQ ID NO:56.
5. recombinant vectors, its each the described RNA of inducing interferential molecule of requirement 1-4 of having the right that is operably connected.
6. composition, it contains the recombinant vectors of each the described RNA of inducing interferential molecule of claim 1-4 or claim 5, and pharmaceutically acceptable carrier.
7. obtain to suppress the method for inducing RNA interferential molecule of epidemic encephalitis B virus, it comprises:
(1), is chosen at the conserved sequence in gene 1 type, 2 types, 3 types and the 4 type strain isolateds, as the potential target sequence according to the whole genome sequence of epidemic encephalitis B virus;
(2) potential target sequence in (1) and human genome database are compared, get rid of and human genome in other encoding sequence or expressed sequence tag homologous sequence, with remaining sequence as target sequence; With
(3) the synthetic RNA interferential molecule of inducing of target sequence that obtains according to step (2).
8. the method for claim 7, wherein said conserved sequence is meant the sequence of guarding more than 80% in the whole genome sequence of gene 1 type, 2 types, 3 types and 4 type strain isolateds, and the sequence of guarding more than 90% in the whole genome sequence of gene 1 type and 3 type strain isolateds is preferably the sequence of guarding more than 95% in gene 1 type and 3 types.
9. the method for claim 7, wherein, the target sequence described in the step (3) is selected from the proteic transcripton sequence of two or more epidemic encephalitis B virus.
10. the method for claim 9, the proteic transcripton sequence of wherein said two or more epidemic encephalitis B virus 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.
11. the recombinant vectors of each the described RNA of inducing interferential molecule of claim 1-4 or claim 5 is used for suppressing the purposes of the composition of epidemic encephalitis B virus genetic expression in preparation.
12. the recombinant vectors of each the described RNA of inducing interferential molecule of claim 1-4 or claim 5 is used for the treatment of and/or prevents purposes in the medicine of epidemic encephalitis type B in preparation.
13. each the described RNA of inducing interferential molecule of claim 1-4 is used for suppressing the purposes of the siRNA of epidemic encephalitis B virus in preparation.
14. suppress epidemic encephalitis B virus siRNA at target sequence, described target sequence is expressed as at least a in the following sequence with cDNA:
(1) arbitrary sequence shown in SEQ ID NO:1～SEQ ID NO:9 and the 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%.
15.siRNA molecule, it contains corresponding with the described target sequence of claim 14 or complementary RNA sequence.
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| CN111304201A (en) * | 2020-02-21 | 2020-06-19 | 华南农业大学 | siRNA for reducing encephalitis B encephalovirus infection and application thereof |
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| CN104805085A (en) * | 2014-01-29 | 2015-07-29 | 江苏命码生物科技有限公司 | Tandem expressed siRNA and use of tandem expressed siRNA in treatment on chronic lymphocytic leukemia |
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| US10273481B2 (en) | 2014-01-29 | 2019-04-30 | Jiangsu Mircromedmark Biotech Co., LTD. | SiRNA in tandem expression and uses thereof in treating chronic lymphocytic leukemia |
| CN106929526A (en) * | 2017-02-17 | 2017-07-07 | 中国人民解放军第四军医大学 | A kind of double luciferase report gene carriers and its construction method and application for screening shRNA |
| CN106929526B (en) * | 2017-02-17 | 2020-06-16 | 中国人民解放军第四军医大学 | Dual-luciferase reporter gene vector for screening shRNA and construction method and application thereof |
| CN111304201A (en) * | 2020-02-21 | 2020-06-19 | 华南农业大学 | siRNA for reducing encephalitis B encephalovirus infection and application thereof |
| CN111304201B (en) * | 2020-02-21 | 2022-10-21 | 华南农业大学 | siRNA for reducing encephalitis B encephalovirus infection and application thereof |
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