CN102181446B - siRNA capability of preventing influenza virus as well as expression vector and pharmaceutical composition of siRNA - Google Patents
siRNA capability of preventing influenza virus as well as expression vector and pharmaceutical composition of siRNA Download PDFInfo
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Abstract
The invention discloses siRNA capability of preventing influenza, the base composition of which is shown in the specification. The invention also provides an expression vector containing the siRNA and a pharmaceutical composition the active ingredient of which is the siRNA or the expression vector. By the siRNA sequence, the reproduction or expression of influenza virus in human bodies or animal bodies can be inhibited.
Description
Technical field
The invention belongs to biomedicine field, specifically relate to a kind of siRNA and expression vector and medicinal compositions of preventing and treating influenza virus.
Background technology
RNA disturbs be found to be life science and the therapeutic treatment of (RNAi) to learn development and bring revolutionary change.SiRNA (siRNA) is that blocking gene is expressed the most efficiently method, is one of kind new medicine of the tool prospect of current pharmacy industry, and fast development and keen competition are just being experienced in relevant research and development of products in the world.The siRNA medicine can and be removed the purpose that disease related gene reaches disease treatment by inhibition.Because its efficient, special targeting and Silencing Mechanisms make it become the jinx of traditional incurable disease.Existing more than 50 kind of siRNA medicine entered clinical front or clinical study by the FDA approval, and the key of development problem of siRNA medicine is the target administration problem, and it has directly restricted the development of siRNA medicine.
In recent years, disturbed relevant research just experiencing volatile growth in academia and biological-pharmacy circle with RNA.Some medicine based on siRNA has now entered clinical stage, comprise the Cand5 that treats moist treating senile maculopathy and ALN-RSV01 (the de Fougerolles A that treats respiratory syncytial virus, Vornlocher HP, Maraganore J, Lieberman J.Interfering with disease:a progress report on siRNA-based therapeutics.Nat Rev Drug Discov2007; 6 (6): 443-53.).To really excavate and realize the treatment potentiality of siRNA, utilize siRNA to capture such as major diseases such as respiratory infectious virus, cancer, central nervous system pathological changes, route of administration and preparation research are that a lot of siRNA drugmaker does not have the final sport technique segment that solves.
Influenza virus is called for short influenza virus, that a kind of mankind of causing and animal suffer from grippal RNA viruses, influenza virus belongs to orthomyxovirus section (Orthomyxoviridae family), the genome of virus is made of 8 single-stranded RNA fragments, 10 protein relevant with virus structure and function of encoding respectively, fragment 1 and 2 encode respectively PB2 and PB1 albumen, fragment 3 coding PA albumen, these three albumen are relevant with viral rna polymerase activity.Fragment 4 coding hemagglutinin glycoprotein (HA), this albumen can combine with the acceptor of human or animal's erythrocyte surface and cause blood coagulation, in the process of virus importing host cell, played the part of the key player, be divided into light chain and heavy chain two portions after the hemagglutinin hydrolysis, the heavy chain part can combine with the sialic acid acceptor on the host cell membrane, and the light chain part can assist peplos and host cell membrane mutually to merge.Fragment 5 coding nucleoprotein (NP), this albumen is combined with genetic material RNA and is formed nucleoglucoprotein body (RNP).Fragment 6 coding NA albumen, this albumen is a tetramer glycoprotein, has the sialic activity of hydrolysis, when the mode of influenza virus through sprouting of maturation breaks away from host cell, the hemagglutinin of virus surface combines with the sialic acid of host cell surface, thereby the virus and host cytolemma is kept in touch, and after HA was hydrolyzed sialic acid, the last contact of cutting off the virus and host cell became a new virus particle.Fragment 7 coding matrix prote m1 and M2, the effect that matter albumen and viral outermost coating are combined closely and played the protection nucleoid and maintain viral space structure.Fragment 8 coding non-structural protein NS 1 and NS2 may be relevant with the genome transcription of virus, and its actual functional capability waits further research.Influenza virus can be divided into four genus according to nucleoprotein (NP) and stromatin (M) wherein: first type (A) B-mode (B), the third type (C) and, the high native Tobamovirus of class holder.A type influenza extensively is present in the mankind and other animals, and its infection scope is wide, is popular form and occurs; Type B exists only in the mankind, often causes the part outburst of influenza; C type influenza is present in human and the pig, and is seldom popular.
Influenza is the most widely one of human infectious disease of popularity, and in China, influenza is one of the most common epidemic disease.Since the recently more than ten years, along with the evolution of influenza virus, new type influenza virus occurs in succession, and 1997, the case of Avian Influenza Virus Infection in Humans class at first occured in Hong Kong; In April, 2009, the novel H1N1 virus that Mexico occurs is propagated and is become worse.In view of the several times flu outbreak that occurs in history, these emerging viruses have caused great threat to the whole mankind's health even life.But existing influenza medicine and influenza vaccines exist very large defective.The siRNA medicine provides effective ways for suppressing influenza virus, siRNA is as the main advantage of anti-virus infection medicine: 1. can select target spot numerous, theoretically, any one encoding gene that needs in the influenza virus life cycle all can be used as the target spot of therapeutic siRNA; The conservative region of the encoding genes such as each albumen all can be used as the target spot of siRNA.2. the R﹠D cycle is short, because the key that siRNA plays a role is the coupling of its functional chain and target sequence, therefore, as long as know in theory the gene order of influenza virus, just can design the siRNA for this sequence, this target sieving and prodrug screening, antibody screening, vaccine antigen than small-molecule drug screens many rapidly.3. pharmaceutical efficacy is high, conservative region design siRNA for a plurality of genes of influenza virus, these siRNA unite the possibility that use can reduce virus variation, escape greatly, are similar to the HAART therapy of HIV treatment, and its effectiveness will be higher than small-molecule drug and the antibody drug of single target spot far away; And according to the calculating of information biology, might design a cover simultaneously for the medicines of a plurality of type influenza viruses.
Summary of the invention
One of purpose of the present invention provides a kind of double-stranded siRNA, and it can suppress influenza virus copying or expressing in people or animal body.
Another object of the present invention provides the expression vector that contains above-mentioned siRNA.
It is the pharmaceutical composition of above-mentioned siRNA sequence or expression vector that another object of the present invention has provided its active ingredient.
The technical scheme that realizes above-mentioned purpose is as follows:
A kind of siRNA that prevents and treats influenza includes at least a among the following siRNA,
SiPB2_001: the positive-sense strand based composition is SEQ ID NO.1, and the antisense strand based composition is SEQ ID NO.2;
SiPB2_002: the positive-sense strand based composition is SEQ ID NO.3, and the antisense strand based composition is SEQ ID NO.4;
SiPB2_003: the positive-sense strand based composition is SEQ ID NO.5, and the antisense strand based composition is SEQ ID NO.6;
SiPB2_004: the positive-sense strand based composition is SEQ ID NO.7, and the antisense strand based composition is SEQ ID NO.8;
SiPB1_001: the positive-sense strand based composition is SEQ ID NO.9, and the antisense strand based composition is SEQ ID NO.10;
SiPB1_002: the positive-sense strand based composition is SEQ ID NO.11, and the antisense strand based composition is SEQ ID NO.12;
SiPA_001: the positive-sense strand based composition is SEQ ID NO.13, and the antisense strand based composition is SEQ ID NO.14;
SiPA_002: the positive-sense strand based composition is SEQ ID NO.15, and the antisense strand based composition is SEQ ID NO.16;
SiPA_003: the positive-sense strand based composition is SEQ ID NO.17, and the antisense strand based composition is SEQ ID NO.18;
SiPA_04: the positive-sense strand based composition is SEQ ID NO.19, and the antisense strand based composition is SEQ ID NO.20;
SiMP_001: the positive-sense strand based composition is SEQ ID NO21, and the antisense strand based composition is SEQ ID NO.22;
SiMP_002: the positive-sense strand based composition is SEQ ID NO.23, and the antisense strand based composition is SEQ ID NO.24;
SiMP_003: the positive-sense strand based composition is SEQ ID NO.25, and the antisense strand based composition is SEQ ID NO.26;
SiMP_004: the positive-sense strand based composition is SEQ ID NO.27, antisense strand based composition SEQ ID NO.28;
SiNP_001: the positive-sense strand based composition is SEQ ID NO.29, and the antisense strand based composition is SEQ ID NO.30;
SiNP_002: the positive-sense strand based composition is SEQ ID NO.31, and the antisense strand based composition is SEQ ID NO.32;
SiNP_003: the positive-sense strand based composition is SEQ ID NO.33, and the antisense strand based composition is SEQ ID NO.34;
SiNS_001: the positive-sense strand based composition is SEQ ID NO.35, and the antisense strand based composition is SEQ ID NO.36;
SiNS_002: the positive-sense strand based composition is SEQ ID NO.37, and the antisense strand based composition is SEQ ID NO.38;
SiNS_003: the positive-sense strand based composition is SEQ ID NO.39, and the antisense strand based composition is SEQ ID NO.40;
SiNS_004: the positive-sense strand based composition is SEQ ID NO.41, and the antisense strand based composition is SEQ ID NO.42;
3 of every chain of above-mentioned siRNA ' is held and also is modified with the base of dangling that any two deoxyribonucleotides form among dA, dT, dG, the dC.
For the establishment influenza of developing wide spectrum, the siRNA of avian influenza virus, we are to collecting to such an extent that more than 3,000 the relevant gene of influenza sorted out, analyzes, compared, therefrom seek out one group of respectively high conserved region territory of target influenza virus PB2, PB1, PA, MP, NP, NS gene, design the siRNAs sequence.
And verify the impact of the modifying method of siRNA with the Luciferase luciferase reporter gene.With synthetic 2 '-O-methylribonucleotide, 2 '-fluorinated pyrimidine nucleosides and lock nucleosides, for a series of siRNA of the synthetic different chemical modification of luciferase gene.Synthetic 2 '-O-methyl or 2 '-fluoro-siRNA molecule are transfected into different cell strain Hela and the MCF-7 cell of stably express luciferase gene by the method for liposome transfection, through certain hour, detect the power of luciferase fluorescence.This result shows, 2 '-O-methyl is different to the reticent influential effect of siRNA for modifying siRNA with F, but not clearly, the pattern of some modification can improve reticent effect, but the reticent effect of siRNA is obviously lowered in the whole 2 '-modification meetings that methylate of antisense strand, and ' siRNA that F modifies is little to reticent influential effect and antisense strand is whole 2.
The invention provides the siRNA that can prevent and treat influenza, one or more combination in the target sequence on the influenza virus gene group of described siRNA and people and animal influenza can suppress influenza virus copying or expressing in human or animal body.At dog kidney passage cell (MDCK), by analyzing virus titer, 50%siRNA is efficient to reach that 60%, 10%siRNA is efficient to be reached more than 85%.
Description of drawings
SiRNA different modifying site figure among Fig. 1, the embodiment 2;
The siRNA of Fig. 2, different modifying is to the interference effect schematic diagram of Luc;
Fig. 3, siRNA disturb behind the 45h restraining effect (100X) schematic diagram to virus;
Behind Fig. 4, the siRNA transfection 68h to the restraining effect schematic diagram of virus;
Fig. 5, siPA various dose disturb behind the 68h restraining effect (200X) schematic diagram to virus;
Fig. 6, specific target siRNA are to preventive and therapeutic effect (X200) schematic diagram of H5N1 type avian influenza virus;
Fig. 7, with the virus titer curve of virus attack time.
Embodiment
SiRNA of the present invention can be used for preparing the pharmaceutical preparation of preventing and treating influenza, and described pharmaceutical preparation can be lipid composition, also can be the composition with form of nanoparticles.Described pharmaceutical preparation is by biocompatiblity molecules and/or biodegradable molecular composition.
SiRNA of the present invention can be in intestines or parenteral admin.Administration is oral administration in the described intestines.Described parenteral admin is administration in administration in administration in administration in intravascular administration, encephalic administration, the pleura, the tumour, intraperitoneal administration, intramuscular administration, lymph administration, the gland, subcutaneous administration, topical, segmental bronchus administration, the tracheae, intranasal administration, inhalation or dropleting medicine-feeding.
By the pharmaceutical preparation of siRNA of the present invention preparation, during application, disposable medicinal concentration in the 0.01mg/kg body weight to the 10mg/kg body weight.
By the expression vector that contains siRNA of the present invention, during application, every dosage is 1X10
5To 1X10
14Individual virion or DNA plasmid vector are with the dosage of 100mg to 4000mg.
Come the present invention is described further below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with describing.But these embodiment only are exemplary.Scope of the present invention is not consisted of any restriction.It will be understood by those skilled in the art that lower without departing from the spirit and scope of the present invention and can make amendment or replace the details of technical solution of the present invention and form, but these modifications and replace and all fall into protection scope of the present invention.
Embodiment one: the siRNA that can prevent and treat influenza
1, the influenza virus target sequence determines
Collect to get more than 3,000 gene that influenza is relevant from the genkbank database, analytical procedure with information biology, these sequences are sorted out, analyze, compared, therefrom seek out one group of respectively high conserved region territory of target influenza virus PB2, PB1, PA, MP, NP, NS gene, thereby find out target sequence, be the high conserved region territory of different plant species influenza virus or influenza virus die mould gene PB2, PB1, PA, MP, NP, NS.
One or several specific effect in selected artificial sequence and the following target sequence can suppress to cause influenza virus gene the copying or expressing in people or animal body of influenza, and described target sequence is as follows:
TARGET?PB2-1：5’GCCATAATTAAGAAGTACA?3’)(SEQ?ID?NO.43)
TARGET?PB2-2：5’GAAGTGCTTACAGGCAACC?3(SEQ?ID?NO.44)
TARGET?PB2-3：5’ACTCTAGCATACTTACTGA?3(SEQIDNO.45)
TARGET?PB2-4：5’GAATTCGGATGGCCATCAA?3(SEQ?IDNO.46)
TARGET?PB1-1：5’GGAACAGGATACACCATGG?3(SEQ?IDNO.47)
TARGET?PB1-2：5’AATGTACCAGAAGTGCTGC?3(SEQ?IDNO.48)
TARGET?PA-1：5’TCCTTTCGTCAGTCCGAGA?3(SEQ?IDNO.49)
TARGET?PA-2：5’TAGAGCCTATGTGGATGGA?3(SEQ?IDNO.50)
TARGET?PA-3：5’AGTGCCTGATTAATGATCC?3(SEQ?ID?NO.51)
TARGET?PA-4：5’TTGCTTAATGCATCTTGGT?3(SEQ?IDNO.52)
TARGET?MP-1：5’GGCTCTCATGGAATGGCTA?3(SEQ?IDNO.53)
TARGET?MP-2：5’AGACAAGACCAATCCTGTC?3(SEQ?IDNO.54)
TARGET?MP-3：5’TGCAGCGTAGACGCTTTGT?3(SEQ?IDNO.55)
TARGET?MP-4：5’TTGCACTTGATATTGTGGA?3(SEQ?IDNO.56)
TARGET?NP-1：5’CACTCACTGAGTGACATCA?3(SEQ?IDNO.57)
TARGET?NP-2：5’ACTGGTGGAGAACGCCAGA?3(SEQ?IDNO.58)
TARGET?NP-3：5’AGGATCTTATTTCTTCGGA?3(SEQ?IDNO.59)
TARGET?NS-1：5’CCATTCCTTGATCGGCTTC?3(SEQ?IDNO.60)
TARGET?NS-2：5’CTTCGCCGAGATCAGAAGT?3(SEQ?IDNO.61)
TARGET?NS-3：5’GGACTTGAATGGAATGATA?3(SEQ?IDNO.62)
TARGET?NS-4：5’GATAACACAGTTCGAGTCT?3(SEQ?IDNO.63)。
2, the relevant siRNA design of virus
For the target sequence design siRNA that finds out, because the high conservative type of target sequence, therefore the siRNA of design has certain wide spectrum effect, can act on multiple influenza virus.These siRNA sequences are as follows:
Sequence title: siPB2_001
SiRNA positive-sense strand: 5 ' GCCAUAAUUAAGAAGUACA dTdG 3 ' (SEQ ID NO.1)
SiRNA antisense strand: 3 ' dGdT CGGUAUUAAUUCUUCAUGU 5 ' (SEQ ID NO.2)
Sequence title: siPB2_002
SiRNA positive-sense strand: 5 ' GAAGUGCUUACAGGCAACC dAdA 3 ' (SEQ ID NO.3)
SiRNA antisense strand: 3 ' dAdA CUUCACGAAUGUCCGUUGG 5 ' (SEQ ID NO.4)
Sequence title: siPB2_003
SiRNA positive-sense strand: 5 ' ACUCUAGCAUACUUACUGA dCdG 3 ' (SEQ ID NO.5)
SiRNA antisense strand: 3 ' dGdC UGAGAUCGUAUGAAUGACU 5 ' (SEQ ID NO.6)
Sequence title: siPB2_004
SiRNA positive-sense strand: 5 ' GAAUUCGGAUGGCCAUCAA dAdA 3 ' (SEQ ID NO.7)
SiRNA antisense strand: 3 ' dAdA CUUAAGCCUACCGGUAGUU 5 ' (SEQ ID NO.8)
Sequence title: siPB1_001
SiRNA positive-sense strand: 5 ' GGAACAGGAUACACCAUGG dCdA 3 ' (SEQ ID NO.9)
SiRNA antisense strand: 3 ' dAdC CCUUGUCCUAUGUGGUACC 5 ' (SEQ ID NO.10)
Sequence title: siPB1_002
SiRNA positive-sense strand: 5 ' AAUGUACCAGAAGUGCUGC dCdA 3 ' (SEQ ID NO.11)
SiRNA antisense strand: 3 ' dAdC UUACAUGGUCUUCACGACG 5 ' (SEQ ID NO.12)
Sequence title: siPA_001
SiRNA positive-sense strand: 5 ' UCCUUUCGUCAGUCCGAGA dAdC 3 ' (SEQ ID NO.13)
SiRNA antisense strand: 3 ' dCdA AGGAAAGCAGUCAGGCUCU 5 ' (SEQ ID NO.14)
Sequence title: siPA_002
SiRNA positive-sense strand: 5 ' UAGAGCCUAUGUGGAUGGA dTdT 3 ' (SEQ ID NO.15)
SiRNA antisense strand: 3 ' dTdT AUCUCGGAUACACCUACCU 5 ' (SEQ ID NO.16)
Sequence title: siPA_003
SiRNA positive-sense strand: 5 ' AGUGCCUGAUUAAUGAUCC dGdG 3 ' (SEQ ID NO.17)
SiRNA antisense strand: 3 ' dGdG UCACGGACUAAUUACUAGG 5 ' (SEQ ID NO.18)
Sequence title: siPA_004
SiRNA positive-sense strand: 5 ' UUGCUUAAUGCAUCUUGGU dTdT 3 ' (SEQ ID NO.19)
SiRNA antisense strand: 3 ' dTdT AACGAAUUACGUAGAACCA 5 ' (SEQ ID NO.20)
Sequence title: siMP_001
SiRNA positive-sense strand: 5 ' GGCUCUCAUGGAAUGGCUA dGdA 3 ' (SEQ ID NO.21)
SiRNA antisense strand: 3 ' dAdG CCGAGAGUACCUUACCGAU 5 ' (SEQ ID NO.22)
Sequence title: siMP_002
SiRNA positive-sense strand: 5 ' AGACAAGACCAAUCCUGUC dAdA 3 ' (SEQ ID NO.23)
SiRNA antisense strand: 3 ' dAdA UCUGUUCUGGUUAGGACAG 5 ' (SEQ ID NO.24)
Sequence title: siMP_003
SiRNA positive-sense strand: 5 ' UGCAGCGUAGACGCUUUGU dAdC 3 ' (SEQ ID NO.25)
SiRNA antisense strand: 3 ' dCdA ACGUCGCAUCUGCGAAACA 5 ' (SEQ ID NO.26)
Sequence title: siMP_004
SiRNA positive-sense strand: 5 ' UUGCACUUGAUAUUGUGGA dTdC 3 ' (SEQ ID NO.27)
SiRNA antisense strand: 3 ' dCdT AACGUGAACUAUAACACCU 5 ' (SEQ ID NO.28)
Sequence title: siNP_001
SiRNA positive-sense strand: 5 ' CACUCACUGAGUGACAUCA dCdT 3 ' (SEQ ID NO.29)
SiRNA antisense strand: 3 ' dTdC GUGAGUGACUCACUGUAGU 5 ' (SEQ ID NO.30)
Sequence title: siNP_002
SiRNA positive-sense strand: 5 ' ACUGGUGGAGAACGCCAGA dAdA 3 ' (SEQ ID NO.31)
SiRNA antisense strand: 3 ' dAdA UGACCACCUCUUGCGGUCU 5 ' (SEQ ID NO.32)
Sequence title: siNP_003
SiRNA positive-sense strand: 5 ' AGGAUCUUAUUUCUUCGGA dGdA 3 ' (SEQ ID NO.33)
SiRNA antisense strand: 3 ' dAdG UCCUAGAAUAAAGAAGCCU 5 ' (SEQ ID NO.34)
Sequence title: siNS_001
SiRNA positive-sense strand: 5 ' CCAUUCCUUGAUCGGCUUC dCdC 3 ' (SEQ ID NO.35)
SiRNA antisense strand: 3 ' dCdC GGUAAGGAACUAGCCGAAG 5 ' (SEQ ID NO.36)
Sequence title: siNS_002
SiRNA positive-sense strand: 5 ' CUUCGCCGAGAUCAGAAGU dGdG 3 ' (SEQ ID NO.37)
SiRNA antisense strand: 3 ' dGdG GAAGCGGCUCUAGUCUUCA 5 ' (SEQ ID NO.38)
Sequence title: siNS_003
SiRNA positive-sense strand: 5 ' GGACUUGAAUGGAAUGAUA dGdA 3 ' (SEQ ID NO.39)
SiRNA antisense strand: 3 ' dAdG CCUGAACUUACCUUACUAU 5 ' (SEQ ID NO.40)
Sequence title: siNS_004
SiRNA positive-sense strand: 5 ' GAUAACACAGUUCGAGUCU dAdT 3 ' (SEQ ID NO.41)
SiRNA antisense strand: 3 ' dTdA CUAUUGUGUCAAGCUCAGA 5 ' (SEQ ID NO.42)
Embodiment two, siRNA modify implementing the impact of interference effect
With synthetic 2 '-O-methylribonucleotide, 2 '-fluorinated pyrimidine nucleosides and lock nucleosides, a series of siRNA for the synthetic different chemical modification of luciferase gene, siRNA sequence is positive-sense strand 5 ' UGAAGAGCCUGAUCAAAUA 3 ', antisense strand is that 3 ' ACUUCUCGGACUAGUUUAU 5 ', the different modifying method in site is such as (Fig. 1).These synthetic and siRNA molecules of modifying are by the method for liposome transfection, be transfected into different cell strain Hela and the MCF-7 cell of stably express luciferase gene, the transfection method summary is for diluting siRNA and liposome with opti-MEM respectively first, after former both are mixed, room temperature leaves standstill 20min, adds to behind liposome siRNA and finishes transfection in the cell.Through certain hour, detect the power of luciferase fluorescence, thus the reticent effect of assessment different modifying siRNA.
This result shows, 2 '-O-methyl is different to the reticent influential effect of siRNA for modifying with F, but is not clearly, and the pattern of some modification can improve reticent effect, and the siRNA that the whole 2 '-F of antisense strand modify is to reticent influential effect not quite (Fig. 2).Used sequence among Fig. 2 is referring to Fig. 1.
The biological activity determination of embodiment three, influenza virus siRNA
Change influenza siRNA over to dog kidney passage cell (MDCK) with the concentration of 100nM, transfection added avian influenza virus H 5 N 1 after 5 hours, and with untransfected virus (normal) and transfection without target-spot siRNA in contrast.After the transfection 45 hours, violet staining detects siRNA to the interference effect of virus.But the result show behind the siRNA viral interference gene establishment its at intracellular proliferation function Fig. 3.With identical method, transfection detects siRNA to the interference effect of virus after 68 hours, referring to Fig. 4, (a upper picture group is 100X to the restraining effect of virus behind the siRNA transfection 68h, next picture group is 200X), after the result showed 68 hours, siRNA still had stronger restraining effect (Fig. 4) to virus.
Embodiment four, different concns siRNA are to viral inhibition
Change influenza siRNA siPA-003 over to dog kidney passage cell (MDCK) with the concentration of 20nM, 100nM, 500nM, transfection added avian influenza virus H 5 N 1 after 5 hours, and with transfection without target-spot siRNA in contrast.After the transfection 68 hours, violet staining detects siRNA to the interference effect of virus.But the result show behind the siRNA viral interference gene establishment its at intracellular proliferation function (Fig. 5).
The biological activity of the biological activity of embodiment five, specific siRNA and siRNA drug combination
Change specific siRNA (siMP-001) and drug combination (siMP-001+siPA-003) over to dog kidney passage cell (MDCK) with 100nM concentration, transfection method is with embodiment two, virus titer is done the hemagglutinin measuring with the chicken erythrocyte, the cellular form violet staining, microscopic examination.Cell image sees that (Fig. 6) and virus titer see (Fig. 7).The result shows copying or expressing of special siRNA energy establishment virus, and Combined Preparation group siRNA is more obvious to the inhibition of virus replication or expression.
Claims (5)
1. siRNA that can prevent and treat influenza, it is characterized in that, described siRNA is: siMP_001: the positive-sense strand based composition is SEQ ID NO.21, the antisense strand based composition is SEQ ID NO.22, and 3 of every chain of above-mentioned siRNA ' is held and also is modified with the base of dangling that any two deoxyribonucleotides form among dA, dT, dG, the dC.
2. the siRNA that can prevent and treat influenza is characterized in that, described siRNA includes: siMP_001: the positive-sense strand based composition is that SEQ ID NO.21 and antisense strand based composition are SEQ ID NO.22, and following at least a,
SiPB2_001: the positive-sense strand based composition is SEQ ID NO.1, and the antisense strand based composition is SEQ ID NO.2;
SiPB2_002: the positive-sense strand based composition is SEQ ID NO.3, and the antisense strand based composition is SEQ ID NO.4;
SiPB2_003: the positive-sense strand based composition is SEQ ID NO.5, and the antisense strand based composition is SEQ ID NO.6;
SiPB2_004: the positive-sense strand based composition is SEQ ID NO.7, and the antisense strand based composition is SEQ ID NO.8;
SiPB1_001: the positive-sense strand based composition is SEQ ID NO.9, and the antisense strand based composition is SEQ ID NO.10;
SiPB1_002: the positive-sense strand based composition is SEQ ID NO.11, and the antisense strand based composition is SEQ ID NO.12;
SiPA_003: the positive-sense strand based composition is SEQ ID NO.17, and the antisense strand based composition is SEQ ID NO.18;
SiMP_002: the positive-sense strand based composition is SEQ ID NO.23, and the antisense strand based composition is SEQ ID NO.24;
SiMP_003: the positive-sense strand based composition is SEQ ID NO.25, and the antisense strand based composition is SEQ ID NO.26;
SiMP_004: the positive-sense strand based composition is SEQ ID NO.27, and the antisense strand based composition is SEQ ID NO.28;
SiNP_001: the positive-sense strand based composition is SEQ ID NO.29, and the antisense strand based composition is SEQ ID NO.30;
SiNP_002: the positive-sense strand based composition is SEQ ID NO.31, and the antisense strand based composition is SEQ ID NO.32;
SiNP_003: the positive-sense strand based composition is SEQ ID NO.33, and the antisense strand based composition is SEQ ID NO.34;
SiNS_001: the positive-sense strand based composition is SEQ ID NO.35, and the antisense strand based composition is SEQ ID NO.36;
SiNS_002: the positive-sense strand based composition is SEQ ID NO.37, and the antisense strand based composition is SEQ ID NO.38;
SiNS_003: the positive-sense strand based composition is SEQ ID NO.39, and the antisense strand based composition is SEQ ID NO.40;
SiNS_004: the positive-sense strand based composition is SEQ ID NO.41, and the antisense strand based composition is SEQ ID NO.42;
3 of every chain of above-mentioned siRNA ' is held and also is modified with the base of dangling that any two deoxyribonucleotides form among dA, dT, dG, the dC.
3. the siRNA that prevents and treats influenza according to claim 2 is characterized in that, described siRNA is the mixing of two kinds of siMP-001 and siPA-003.
4. the expression vector that has each described siRNA of claim 1-3.
5. the pharmaceutical composition that can prevent and treat influenza is characterized in that, its active ingredient is each described siRNA of claim 1-3 or expression vector claimed in claim 4.
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