CN103667285A - SiRNA (small interfering Ribose Nucleic Acid) capable of preventing and controlling influenza, and pharmaceutical composition and medical application thereof - Google Patents
SiRNA (small interfering Ribose Nucleic Acid) capable of preventing and controlling influenza, and pharmaceutical composition and medical application thereof Download PDFInfo
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Abstract
The invention discloses a siRNA (small interfering Ribose Nucleic Acid) of targeting genes ST6GAL1 and ST3GAL4 capable of preventing and controlling influenza. The siRNA comprises at least one of siST6GAL1_001, siST6GAL1_002, siST3GAL4_001, and siST3GAL4_002. The invention also provides a pharmaceutical composition of which the siRNA serves as an effective ingredient. The invention also provides an application of the siRNA in preparation of the pharmaceutical composition, a medicine and a prevention preparation for preventing and controlling influenza virus. By adopting the pharmaceutical composition, medicine and prevention preparation, the expression of a human influenza A virus sialic acid receptor and an avian influenza A virus sialic acid receptor on the surface of epithelial cell of human respiratory tract can be inhibited, thus the adsorption and infection of human and avian influenza A virus can be reduced effectively; in addition, the effective rate of the siRNA reaches more than 80% in the respiratory tract epithelial cell line.
Description
Technical field
The present invention relates to medical technical field, relate in particular to a kind of siRNA that prevents and treats influenza and pharmaceutical composition thereof, medicinal use.
Background technology
Influenza virus is called for short influenza virus, is that a kind of mankind of causing and animal suffer from grippal RNA viruses; Influenza virus can be divided into four genus according to nucleoprotein (NP) and stromatin (M) wherein: the high native Tobamovirus of A type (A), B-mode (B), the third type (C) and class holder.Influenza A is extensively present in the mankind and other animals, and its infection scope is wide, is popular form and occurs, endangers also the most serious; B-modely exist only in the mankind, often cause the part outburst of influenza; Influenza C is present in the mankind and pig, seldom popular.
Influenza virus mainly enters in host by infecting human airway epithelium cell, causes various constitutional symptoms, even serious complication! The sialic acid molecule on airway epithelial cell surface is as the acceptor of influenza virus absorption and closely related with influenza infection.The influenza virus in people source is partial to identify α-2, and 6-sialic acid links as acceptor, and the influenza virus in fowl source is partial to α-2, and 3-sialic acid links as acceptor.
Sialic acid molecule is linked at the glycoprotein of cell surface and the end of glycolipid, in each group biological processes (as immunogen identification, virus infection and cell absorption etc.), plays an important role.Sialytransferase is the enzyme of the sialic acid molecule content of the main adjusting cell surface of a class.The about kind more than 20 of Human genome group coding relates to glycoprotein and the sialylated sialytransferase of glycolipid.Up-to-date studies confirm that, beta galactose α-2,6-sialytransferase I(ST6GAL I) be responsible for α-2,6-sialic acid is added to GAL β 1-4GlcNAc(disaccharides of N-link and some O-link of glycoprotein analog glycan) on; And beta galactose α-2,3-sialytransferase IV(ST3GAL IV) be mainly responsible for α-2,3-sialic acid is added on the N-link of glycoprotein analog glycan and the GAL β 1-4GlcNAc of some O-link.The gene of coding ST6GAL I and ST3GAL IV is at human airway high expression level, and this points out these two kinds of enzymes relevant to the mechanism of influenza virus main infection human airway, and likely becomes the target spot of prevention and treatment influenza virus.Current published about in research, there is not yet and utilize the siRNA for preparing target ST6GAL1 and ST3GAL4 gene for suppressing multiple influenza virus sub-strain, especially reduce influenza virus absorption and the report of minimizing infection.
SiRNA (siRNA) is the double-stranded RNA that a class length is about 21bp, when it is imported into after cell, can mediate the mRNA degraded corresponding with its sequence, thereby play specificity, suppresses expression of target gene, and this phenomenon is called RNA interference mechanism.In recent years, the develop rapidly of RNA perturbation technique, be widely used in the every field of life science, comprise antiviral therapy aspect, for example, the siRNA that has gene (comprising HIV, poliovirus, hepatitis C virus, west Nile virus and influenza virus) the design special target studies confirm that for each viroid can play effective antivirus action in vivo and in vitro.But the siRNA that gene necessary for influenza infection, host itself designs carries out less report of antiviral research.
The antiviral that can be used at present treatment and flu-prevention virus mainly contains neuraminidase inhibitor (NAI), as Oseltamivir and zanamivir; And M2 inhibitors of ion channels diamantane amine, comprise amantadine and Rimantadine.But above two class combination drugs have various side effects and limitation, as easy generation resistance etc., this also becomes the major issue of clinical treatment and new drug development.
Eventually the above, researching and developing novel specificity Tamiflu higher, that be difficult for producing resistance becomes problem demanding prompt solution in the industry.Meanwhile, for the necessary gene of influenza infection, design high specificity target medicinal, and study and apply also become in the industry in the urgent need to.
Summary of the invention
In order to solve the problems of the technologies described above, the present invention designs the siRNA of target ST6GAL1 and ST3GAL4 gene, defines its antiviral effect and mechanism of action thereof, for the siRNA anti-influenza virus medicament of the targeting of development of new provides basic data.
One of object of the present invention is to provide a kind of double-stranded siRNA, and it can suppress the expression of the people source influenza A virus sialic acid acceptor (SAa2,6GAL) of human respiratory surface epithelial cell, thereby reduces absorption and the infection of people source influenza A virus.
Another object of the present invention is to provide a kind of double-stranded siRNA, and it can suppress the expression of the fowl source influenza A virus sialic acid acceptor (SAa2,3GAL) of human respiratory surface epithelial cell, thereby reduces absorption and the infection of fowl source influenza A virus.
Another object of the present invention has been to provide the pharmaceutical composition that its active ingredient is above-mentioned siRNA sequence, suppresses to infect for reducing the absorption of multiple influenza virus sub-strain.
Another object of the present invention has been to provide the medicinal use of described siRNA, for the preparation of pharmaceutical composition, medicine and the preventing preparation that can prevent and treat influenza virus.
For foregoing invention object, the invention provides following technical scheme:
The invention provides the siRNA of target ST6GAL1 and ST3GAL4 gene, described siRNA be following at least one:
SiST6GAL1_001: positive-sense strand based composition is SEQ ID NO.1, antisense strand based composition is SEQ ID NO.2;
SiST6GAL1_002: positive-sense strand based composition is SEQ ID NO.3, antisense strand based composition is SEQ ID NO.4;
SiST3GAL4_001: positive-sense strand based composition is SEQ ID NO.5, antisense strand based composition is SEQ ID NO.6;
SiST3GAL4_002: positive-sense strand based composition is SEQ ID NO.7, antisense strand based composition is SEQ ID NO.8.
As the improvement of such scheme, 3 ' the terminal modified bases of dangling of every chain of described siRNA.
The chain of described siRNA comprises positive-sense strand and antisense strand.
As the improvement of such scheme, described in the base any one or more deoxyribonucleotides in dA, dT, dG, dC that dangle form.
As the improvement of such scheme, described in base any two deoxyribonucleotides in dA, dT, dG, dC that dangle form.
As the improvement of such scheme, described siRNA is through any one or more modification in phosphorylation, methoxylation, sulfo-, cholesterol, Cy3, Cy5, vitamin H, VITAMIN, folic acid, cholic acid, PEG.
As the improvement of such scheme, described siRNA also process is the modification of 2 '-O-methylribonucleotide and/or 2 '-fluoro.
Improvement as such scheme, described siRNA is the mixing of two kinds of siST6GAL1_001 and siST3GAL4_001, the mixing that siST6GAL1_001 and siST3GAL4_002 are two kinds, the mixing that siST6GAL1_002 and siST3GAL4_001 are two kinds, or the mixing of two kinds of siST6GAL1_002 and siST3GAL4_002.
Correspondingly, the present invention also provides a kind of pharmaceutical composition of preventing and treating influenza, and its active ingredient is above-mentioned siRNA.
Correspondingly, the present invention also provides the medicinal use of above-mentioned siRNA, for the preparation of pharmaceutical composition, medicine and the preventing preparation that can prevent and treat influenza virus.
Implement the embodiment of the present invention, there is following beneficial effect:
The invention provides and can prevent and treat the target ST6GAL1 of influenza and the siRNA of ST3GAL4 gene, the people source influenza A virus sialic acid acceptor (SAa2 that can suppress human respiratory surface epithelial cell, 6GAL) and fowl source influenza A virus sialic acid acceptor (SAa2, expression 3GAL), thus absorption and the infection of people source and fowl source influenza A virus can effectively be reduced.In airway epithelial cell system (A549, HBE and HEp-2), by analyzing virus titer, described siRNA is efficient to be reached more than 80%.
The present invention also provides the pharmaceutical composition that can prevent and treat influenza, and its activeconstituents is the siRNA of target ST6GAL1 and ST3GAL4 gene, for suppressing the absorption of multiple influenza virus sub-strain, reduces infection.
The present invention also provides the application of the siRNA of target ST6GAL1 and ST3GAL4 gene, especially the application in preparing pharmaceutical composition, medicine and the preventing preparation that can prevent and treat influenza virus.SiRNA class antiviral drug compositions of the present invention, medicine and preventing preparation have high, the difficult features such as resistance that produce of specificity.And can coordinate special carrier, and can pass through respiratory tract inhalation, at treatment respiratory viral infection disease, there is unique advantage.
Accompanying drawing explanation
Figure 1A and Figure 1B are that real-time quantitative RT-PCR detects the suppression efficiency figure of described siRNA to ST6GAL1 and ST3GAL4 gene;
Fig. 2 A and Fig. 2 B are that western blot detects the suppression efficiency figure of described siRNA to sialytransferase ST6GAL Ι and ST3GAL IV expression at protein level;
Fig. 3 A, Fig. 3 B, Fig. 3 C, Fig. 3 D, Fig. 3 E and Fig. 3 F are that immunofluorescence detects the restraining effect design sketch of described siRNA to cell surface sialic acid acceptor molecule;
Fig. 4 A and Fig. 4 B are the restraining effect design sketchs that siRNA expresses cell surface sialic acid acceptor molecule described in Flow cytometry;
Fig. 5 is that MTT test detects the affect schematic diagram of described siRNA on cell normal growth curve;
Fig. 6 A, Fig. 6 B, Fig. 6 C, Fig. 6 D, Fig. 6 E and Fig. 6 F are that immunofluorescence detects the effect schematic diagram that described siRNA reduces influenza virus adherent cell surface;
Fig. 7 A, Fig. 7 B are that quantitative RT-PCR detect to infect described siRNA in 4 hours and reduces the schematic diagram that enters the influenza virus gene quantity in transfectional cell;
Fig. 8 A, Fig. 8 B are the restraining effect schematic diagram that the many replicative cycles of described siRNA infected by influenza infect airway epithelial;
Fig. 9 A, Fig. 9 B are the restraining effect schematic diagram that the pharmaceutical composition of described siRNA sequence infects multiple influenza virus sub-strain;
Figure 10 is that ELISA detects described siRNA in the result schematic diagram of the induction INF-of airway epithelial cell system alpha expression.
Embodiment
For making the object, technical solutions and advantages of the present invention clearer, below in conjunction with accompanying drawing, the present invention is described in further detail.
The invention belongs to medical technical field, particularly, the present invention relates to a kind of siRNA and pharmaceutical composition thereof of preventing and treating influenza virus.The invention still further relates to described siRNA in the medicine of pharmaceutical compositions, resisiting influenza virus, the purposes of preventing preparation.The invention still further relates to the method for the treatment disease that causes of influenza virus and disease, obstacle or the illness concurrent with it.
For designing the siRNA of efficient target ST6GAL1 and ST3GAL4 gene, we analyze the sequence of the upper ST6GAL1 of GenBank and ST3GAL4 gene, design candidate siRNA synthetic.Then apply real-time quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction, reverse transcription PCR), western blot(western blotting), the method such as immunofluorescence and flow cytometry optimizes efficiently, the siRNA of high specific.
By importing candidate siRNA, to airway epithelial cell, be, employment source Ji Qin source influenza infection then, experimental result determines that described siRNA reduces viruses adsorption and infection by the expression of the sialic acid acceptor molecule of inhibition cell surface.In airway epithelial cell system (A549, HBE and Hep-2), by analyzing virus titer, described siRNA is efficient to be reached more than 80%.
SiRNA of the present invention can be used for the pharmaceutical preparation that preparation prevents and treats influenza, and described pharmaceutical preparation can be lipid composition, can be also 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 through parenteral admin.Described parenteral admin is administration in topical, segmental bronchus administration, tracheae, intranasal administration, inhalation or dropleting medicine-feeding.
Below in conjunction with specific embodiment, the present invention is described further, advantage and disadvantage of the present invention will be more clear along with describing.But these embodiment are only exemplary.Scope of the present invention is not formed to 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 the details of technical solution of the present invention and form be modified or be replaced, but these modifications and replacement all fall into protection scope of the present invention.
Embodiment mono-: the screening of target ST6GAL1 and ST3GAL4 gene siRNA
From GenBank database lookup to ST6GAL1 with ST3GAL4 gene order, and determine suitable target sequence by bioinformatics technique; For the target sequence design siRNA finding out, these siRNA sequences are as follows:
Sequence title: siST6GAL1_001
SiRNA positive-sense strand: 5 ' CAGCCAACUUCCAACAAGAdTdT 3 ' (SEQ ID NO.1)
SiRNA antisense strand: 3 ' dTdT GUCGGUUGAAGGUUGUUCU 5 ' (SEQ ID NO.2)
Sequence title: siST6GAL1_002
SiRNA positive-sense strand: 5 ' GUACCAGAAUCCGGAUUAU dTdT 3 ' (SEQ ID NO.3)
SiRNA antisense strand: 3 ' dTdT CAUGGUCUUAGGCCUAAUA 5 ' (SEQ ID NO.4)
Sequence title: siST3GAL4_001
SiRNA positive-sense strand: 5 ' GGUCAUCAGAUUGAACAAU dTdT 3 ' (SEQ ID NO.5)
SiRNA antisense strand: 3 ' dTdT CCAGUAGUCUAACUUGUUA 5 ' (SEQ ID NO.6)
Sequence title: siST3GAL4_002
SiRNA positive-sense strand: 5 ' GCAGACCAUUCACUACUAU dTdT 3 ' (SEQ ID NO.7)
SiRNA antisense strand: 3 ' dTdT CGUCUGGUAAGUGAUGAUA 5 ' (SEQ ID NO.8)
Embodiment bis-, the described siRNA suppression efficiency to ST6GAL1 and ST3GAL4 genetic expression
These synthesize and the siRNA molecule modified by the method for liposome transfection, be transfected into representative airway epithelial cell strain A549, HBE and HEp-2 cell, transfection method summary is for first diluting siRNA and liposome with Opti-MEM respectively, then both are mixed, the standing 20min of room temperature adds to and in cell, completes transfection after liposome siRNA.After 48 hours, adopt real-time quantitative RT-PCR detection and western blot at protein level, to detect the expression of ST6GAL1 and ST3GAL4 gene, thereby assess the reticent effect (Figure 1A and Figure 1B, Fig. 2 A and Fig. 2 B) of different siRNA.
Result shows: the ST6GAL1 of airway epithelial cell and the relative expression quantity of ST3GAL4 of transfection target siRNA obviously reduce, described siRNA to the suppression efficiency of target gene all more than 80%.Specific as follows:
Figure 1A is that real-time quantitative RT-PCR detects the suppression efficiency figure of described siRNA to ST6GAL1 gene.Figure 1A has shown candidate siRNA(si-ST6GAL1-001; Si-ST6GAL1-002; Si-ST6GAL1-003; Si-ST6GAL1-004), transfection is without target-spot siRNA (Non-specific siRNA) and Normal group (Normal cells) relative expression quantity to ST6GAL1 gene in A549, HBE and tri-kinds of airway epithelial cells systems of HEp-2 respectively.The result being shown from Figure 1A, the ST6GAL1 relative expression quantity of the airway epithelial cell of candidate's transfection siRNA obviously reduces, described siRNA to the suppression efficiency of target gene all more than 80%.
It should be noted that, the candidate siRNA in Figure 1A and transfection have significant difference ， ﹡ to the relative expression quantity of ST6GAL1 gene without target-spot siRNA in three kinds of airway epithelial cell systems
p< 0.05.
Figure 1B is that real-time quantitative RT-PCR detects the suppression efficiency figure of described siRNA to ST3GAL4 gene.Figure 1B has shown candidate siRNA(si-ST3GAL4-001; Si-ST3GAL4-002; Si-ST3GAL4-003; Si-ST3GAL4-004), transfection is without target-spot siRNA (Non-specific siRNA) and Normal group (Normal cells) relative expression quantity of ST3GAL4 gene in A549, HBE and tri-kinds of airway epithelial cells systems of HEp-2 respectively.The result being shown from Figure 1B, the ST3GAL4 relative expression quantity of the airway epithelial cell of candidate's transfection siRNA obviously reduces, described siRNA to the suppression efficiency of target gene all more than 80%.
It should be noted that, the candidate siRNA in Figure 1B and transfection have significant difference ， ﹡ to the relative expression quantity of ST3GAL4 gene without target-spot siRNA in three kinds of airway epithelial cell systems
p< 0.05.
Fig. 2 A is that western blot detects at protein level the suppression efficiency figure that described siRNA expresses sialytransferase ST6GAL Ι.Fig. 2 A has shown, take β-Actin as reference, candidate siRNA(si-ST6GAL1-001; While si-ST6GAL1-002) disturbing 48h, the expression amount of sialytransferase ST6GAL Ι obviously reduces.
Wherein, 1 of Fig. 2 A, 2,3,4 experimental group represent respectively: 1, blank (Blank control); 2, transfection is without target-spot siRNA (Non-specific siRNA); 3, si-ST6GAL1-001; 4, si-ST6GAL1-002.
Fig. 2 B is that western blot detects at protein level the suppression efficiency figure that described siRNA expresses sialytransferase ST3GAL IV.Fig. 2 B has shown, take β-Actin as reference, candidate siRNA(si-ST3GAL4-001; While si-ST3GAL4-002) disturbing 48h, the expression amount of sialytransferase ST3GAL IV obviously reduces.
Wherein, 1 of Fig. 2 B, 2,3,4 experimental group represent respectively: 1, blank (Blank control); 2, transfection is without target-spot siRNA (Non-specific siRNA); 3, si-ST3GAL4-001; 4, si-ST3GAL4-002.
Also it should be noted that, Opti-MEM is improved Eagle MEM substratum, contains HEPES(4-hydroxyethyl piperazine ethanesulfonic acid; And added xanthoglobulin, thymus pyrimidine, Sodium.alpha.-ketopropionate, L-glutaminate, trace element and somatomedin N-(2-hydroxyethyl) piperazine-N'-2-ethane sulfonic acid) and sodium bicarbonate buffer liquid.Phenol red concentration is low to moderate 1.1 mg/L.
The restraining effect that embodiment tri-, described siRNA express cell surface sialic acid acceptor molecule
Described siRNA is imported to airway epithelial cell, and method is with embodiment after bis-, 56 hours, immunofluorescence and the restraining effect of Flow cytometry siRNA to cell surface sialic acid acceptor molecule.As Fig. 3 A, Fig. 3 B, Fig. 3 C, Fig. 3 D, Fig. 3 E, Fig. 3 F, and shown in Fig. 4 A, Fig. 4 B, result shows, the have obvious restraining effect of siRNA to the expression of cell surface sialic acid acceptor molecule, and suppression efficiency is all more than 80%, specific as follows:
Fig. 3 A be immunofluorescence detect untransfected target siRNA and transfection target ST6GAL1--siRNA respectively to HBE cell surface sialic acid acceptor molecule SAa2, the restraining effect design sketch of 6GAL.In Fig. 3 A, one of left side classifies untransfected target siRNA as to HBE cell surface sialic acid acceptor molecule SAa2, the restraining effect design sketch of 6GAL, and right side one classify transfection as target ST6GAL1--siRNA cell to HBE cell surface sialic acid acceptor molecule SAa2, the restraining effect design sketch of 6GAL.Under laser confocal microscope, observe: more SAa2 is seen, the expression of 6GAL in HBE cell (left side) surface of untransfected target siRNA; And transfection the SAa2 on HBE cell (right side) surface of target ST6GAL1--siRNA, 6GAL obviously reduces.
The result being shown from Fig. 3 A, transfection target ST6GAL1--siRNA to HBE cell surface sialic acid acceptor molecule SAa2,6GAL expresses obvious restraining effect, suppression efficiency is all more than 80%.
In Fig. 3 A, respectively organize accompanying drawing and adopt following mark mode, a, SNA-FITC, b, DAPI, c, Merge; D, SNA-FITC, e, DAPI, f, Merge.Specific as follows:
(1) a, d group adopts fluorescent mark phytohemagglutinin SNA-FITC mark, adopt the Williams Elder Twig lectin of the marked by fluorescein isothiocyanate of specific recognition sialic acid acceptor to act on and respectively organize cell, after detecting transfection by flow cytometer, respectively organize cell surface SAa2,6GAL content, with average fluorescent strength fluorescence intensity, MFI represents cell surface SAa2,6GAL content.
It should be noted that, biological lectin Sambucus Nigra Lectin(SNA) can be specifically and the SAa2 of cell surface, 6GAL combination, thus play the effect of molecular probe, so adopt the SNA of FITC mark to detect the airway epithelial cell through candidate siRNA transfection or untransfected.
(2) b, e group have adopted DAPI mark, and DAPI is 4', 6-diamidino-2-phenylindone (4', 6-diamidino-2-phenylindole), be a kind of can with the fluorescence dye of the powerful combination of DNA, can be used for the dyeing of viable cell and fixed cell core.
(3) c, f group have adopted Merge mark, and c group fluorescent mark picture is the merging stack of a group fluorescent mark picture and b group fluorescent mark picture, and f group fluorescent mark picture is the merging stack of d group fluorescent mark picture and e group fluorescent mark picture.
Fig. 3 B be immunofluorescence detect untransfected target siRNA and transfection target ST3GAL4--siRNA respectively to HBE cell surface sialic acid acceptor molecule SAa2, the restraining effect design sketch of 3GAL.In Fig. 3 B, one of left side classifies untransfected target siRNA as to HBE cell surface sialic acid acceptor molecule SAa2, the restraining effect design sketch of 3GAL, and right side one classify transfection as target ST3GAL4--siRNA cell to HBE cell surface sialic acid acceptor molecule SAa2, the restraining effect design sketch of 3GAL.Under laser confocal microscope, observe: more SAa2 is seen, the expression of 3GAL in HBE cell (left side) surface of untransfected target siRNA; And transfection the SAa2 on HBE cell (right side) surface of target ST3GAL4--siRNA, 3GAL obviously reduces.
The result being shown from Fig. 3 B, transfection target ST3GAL4--siRNA to HBE cell surface sialic acid acceptor molecule SAa2,3GAL expresses obvious restraining effect, suppression efficiency is all more than 80%.
In Fig. 3 B, respectively organize accompanying drawing and adopt following mark mode, a, MAA-FITC, b, DAPI, c, Merge; D, MAA-FITC, e, DAPI, f, Merge.Specific as follows:
(1) a, d group adopts fluorescent mark phytohemagglutinin MAA-FITC mark, after detecting transfection by flow cytometer, respectively organize cell surface SAa2,3GAL content, with average fluorescent strength fluorescence intensity, MFI represents cell surface SAa2,3GAL content.
It should be noted that, biological lectin Maackia Amurensis Lectin(MAA) can be specifically and the SAa2 of cell surface, 3GAL combination, thus play the effect of molecular probe, so adopt the MAA of FITC mark to detect the airway epithelial cell through candidate siRNA transfection or untransfected.
(2) b, e group have adopted DAPI mark, and DAPI is 4', 6-diamidino-2-phenylindone (4', 6-diamidino-2-phenylindole), be a kind of can with the fluorescence dye of the powerful combination of DNA, can be used for the dyeing of viable cell and fixed cell core.
(3) c, f group have adopted Merge mark, and c group fluorescent mark picture is the merging stack of a group fluorescent mark picture and b group fluorescent mark picture, and f group fluorescent mark picture is the merging stack of d group fluorescent mark picture and e group fluorescent mark picture.
Fig. 3 C be immunofluorescence detect untransfected target siRNA and transfection target ST6GAL1--siRNA respectively to HEp-2 cell surface sialic acid acceptor molecule SAa2, the restraining effect design sketch of 6GAL.In Fig. 3 C, one of left side classifies untransfected target siRNA as to HEp-2 cell surface sialic acid acceptor molecule SAa2, the restraining effect design sketch of 6GAL, and right side one classify transfection as target ST6GAL1--siRNA cell to HEp-2 cell surface sialic acid acceptor molecule SAa2, the restraining effect design sketch of 6GAL.Under laser confocal microscope, observe: more SAa2 is seen, the expression of 6GALl in HEp-2 cell (left side) surface of untransfected target siRNA; And transfection the SAa2 on HEp-2 cell (right side) surface of target ST6GAL1--siRNA, 6GAL obviously reduces.
The result being shown from Fig. 3 C, transfection target ST6GAL1--siRNA to HEp-2 cell surface sialic acid acceptor molecule SAa2,6GAL expresses obvious restraining effect, suppression efficiency is all more than 80%.
In Fig. 3 C, respectively organize accompanying drawing and adopt following mark mode, a, SNA-FITC, b, DAPI, c, Merge; D, SNA-FITC, e, DAPI, f, Merge.Further, in Fig. 3 C: a, d group, b, e group, the mark mode of c, f group is identical with Fig. 3 A, does not repeat them here.
Fig. 3 D be immunofluorescence detect untransfected target siRNA and transfection target ST3GAL4--siRNA respectively to HEp-2 cell surface sialic acid acceptor molecule SAa2, the restraining effect design sketch of 3GAL.In Fig. 3 D, one of left side classifies untransfected target siRNA as to HEp-2 cell surface sialic acid acceptor molecule SAa2, the restraining effect design sketch of 3GAL, and right side one classify transfection as target ST3GAL4--siRNA cell to HEp-2 cell surface sialic acid acceptor molecule SAa2, the restraining effect design sketch of 3GAL.Under laser confocal microscope, observe: more SAa2 is seen, the expression of 3GAL in HEp-2 cell (left side) surface of untransfected target siRNA; And transfection the SAa2 on HEp-2 cell (right side) surface of target ST3GAL4--siRNA, 3GAL obviously reduces.
The result being shown from Fig. 3 D, transfection target ST3GAL4--siRNA to HEp-2 cell surface sialic acid acceptor molecule SAa2,3GAL expresses obvious restraining effect, suppression efficiency is all more than 80%.
In Fig. 3 D, respectively organize accompanying drawing and adopt following mark mode, a, MAA-FITC, b, DAPI, c, Merge; D, MAA-FITC, e, DAPI, f, Merge.Further, in Fig. 3 D: a, d group, b, e group, the mark mode of c, f group is identical with Fig. 3 B, does not repeat them here.
Fig. 3 E be immunofluorescence detect untransfected target siRNA and transfection target ST6GAL1--siRNA respectively to A549 cell surface sialic acid acceptor molecule SAa2, the restraining effect design sketch of 6GAL.In Fig. 3 E, one of left side classifies untransfected target siRNA as to A549 cell surface sialic acid acceptor molecule SAa2, the restraining effect design sketch of 6GAL, and right side one classify transfection as target ST6GAL1--siRNA cell to A549 cell surface sialic acid acceptor molecule SAa2, the restraining effect design sketch of 6GAL.Under laser confocal microscope, observe: more SAa2 is seen, the expression of 6GAL in A549 cell (left side) surface of untransfected target siRNA; And transfection the SAa2 on A549 cell (right side) surface of target ST6GAL1--siRNA, 6GAL obviously reduces.
The result being shown from Fig. 3 E, transfection target ST6GAL1--siRNA to A549 cell surface sialic acid acceptor molecule SAa2,6GAL expresses obvious restraining effect, suppression efficiency is all more than 80%.
In Fig. 3 E, respectively organize accompanying drawing and adopt following mark mode, a, SNA-FITC, b, DAPI, c, Merge; D, SNA-FITC, e, DAPI, f, Merge.Further, in Fig. 3 E: a, d group, b, e group, the mark mode of c, f group is identical with Fig. 3 A, does not repeat them here.
Fig. 3 F be immunofluorescence detect untransfected target siRNA and transfection target ST3GAL4--siRNA respectively to A549 cell surface sialic acid acceptor molecule SAa2, the restraining effect design sketch of 3GAL.In Fig. 3 F, one of left side classifies untransfected target siRNA as to A549 cell surface sialic acid acceptor molecule SAa2, the restraining effect design sketch of 3GAL, and right side one classify transfection as target ST3GAL4--siRNA cell to A549 cell surface sialic acid acceptor molecule SAa2, the restraining effect design sketch of 3GAL.Under laser confocal microscope, observe: more SAa2 is seen, the expression of 3GAL in A549 cell (left side) surface of untransfected target siRNA; And transfection the SAa2 on A549 cell (right side) surface of target ST3GAL4--siRNA, 3GAL obviously reduces.
The result being shown from Fig. 3 F, transfection target ST3GAL4--siRNA to A549 cell surface sialic acid acceptor molecule SAa2,3GAL expresses obvious restraining effect, suppression efficiency is all more than 80%.
In Fig. 3 F, respectively organize accompanying drawing and adopt following mark mode, a, MAA-FITC, b, DAPI, c, Merge; D, MAA-FITC, e, DAPI, f, Merge.Further, in Fig. 3 F: a, d group, b, e group, the mark mode of c, f group is identical with Fig. 3 B, does not repeat them here.
Fig. 4 A is that Flow cytometry Normal group and target siRNA treatment group are to cell surface sialic acid acceptor molecule SAa2, the restraining effect design sketch of 6GAL.Fig. 4 A has shown the SAa2 of flow cytometry detection by quantitative cell surface, the expression of 6GAL acceptor, left side is Normal group, average fluorescent strength (MFI=110.47 ± 6.28), and the average fluorescent strength of right side target siRNA treatment group obviously lowers (MFI=21.35 ± 3.002).
The result being shown from Fig. 4 A, target siRNA treatment group is to cell surface sialic acid acceptor molecule SAa2, and 6GAL has obvious restraining effect, and suppression efficiency is all more than 80%.
It should be noted that, Fig. 4 A adopts fluorescent mark phytohemagglutinin SNA-FITC mark.
Fig. 4 B is that Flow cytometry Normal group and target siRNA treatment group are to cell surface sialic acid acceptor molecule SAa2, the restraining effect design sketch of 3GAL.Fig. 4 B has shown the SAa2 of flow cytometry detection by quantitative cell surface, the expression of 3GAL acceptor, left side is Normal group, average fluorescent strength (MFI=120.69 ± 10.68), and the average fluorescent strength of right side target siRNA treatment group obviously lowers (MFI=25.81 ± 4.62)
The result being shown from Fig. 4 B, target siRNA treatment group is to cell surface sialic acid acceptor molecule SAa2, and 3GAL has obvious restraining effect, and suppression efficiency is all more than 80%.
It should be noted that, Fig. 4 B adopts fluorescent mark phytohemagglutinin MAA-FITC mark.
In sum, the result of Fig. 3 and Fig. 4 shows, the have obvious restraining effect of the siRNA of target ST6GAL1 and ST3GAL4 gene to the expression of cell surface sialic acid acceptor molecule, and suppression efficiency is all more than 80%.
Embodiment tetra-, the impact of detection siRNA on cell normal growth
Described siRNA is imported to airway epithelial cell, and method is with embodiment bis-, and application MTT test detects siRNA to the impact of the growth curve of cell (as shown in Figure 5).Result shows, in the effective concentration (5nM) of siRNA, the growth of cell is had no significant effect.
The impact on embodiment five, siRNA infected by influenza adherent cell surface
Described siRNA is imported to airway epithelial cell, and method is with embodiment bis-, transfection after 72 hours, join in the stream of people Influenza Virus H1N1 and avian influenza virus H9N2, and with non-transfected cells and transfection without target-spot siRNA in contrast.After 2 hours, immunofluorescence detected result shows: the virion of described siRNA treatment group cell surface absorption obviously reduces (as shown in Figure 6).
Fig. 6 A is that immunofluorescence detects the effect schematic diagram of described siRNA to new first stream H1N1 influenza virus absorption A549 cell surface.In Fig. 6 A, one of left side classifies the effect schematic diagram of the non-targeted siRNA of transfection to new first stream H1N1 influenza virus absorption A549 cell surface as, and a siRNA cell of classifying transfection target ST6GAL1 gene as on right side adsorbs the effect schematic diagram of A549 cell surface to new first stream H1N1 influenza virus.
Under laser confocal microscope, observe: a large amount of virion absorption is seen on new first stream H1N1 influenza virus absorption A549 cell (left side) surface of the non-targeted siRNA of transfection; And the seldom virion absorption of amount is only seen on new first stream H1N1 influenza virus absorption A549 cell (right side) surface of the siRNA of transfection target ST6GAL1 gene.
The result being shown from Fig. 6 A, the siRNA of target ST6GAL1 gene has obvious restraining effect to new first stream H1N1 influenza virus absorption A549 cell surface.Compare without target-spot siRNA with non-transfected cells and transfection, the virion of the siRNA treatment group cell surface absorption of target ST6GAL1 gene obviously reduces.
In Fig. 6 A, respectively organize accompanying drawing and adopt following mark mode: a, Anti-HA, b, DAPI, c, Merge, d, Zoom; E, Anti-HA, f, DAPI, g, Merge, h, Zoom, specific as follows:
(1) a, e group adopts resisiting influenza virus hemagglutinin antibody (Anti-HA) mark, does immunofluorescence experiment.Anti-HA is highly purified mouse monoclonal antibody.
(2) b, f group have adopted DAPI mark, and DAPI is 4', 6-diamidino-2-phenylindone (4', 6-diamidino-2-phenylindole), be a kind of can with the fluorescence dye of the powerful combination of DNA, can be used for the dyeing of viable cell and fixed cell core.
(3) c, g group have adopted Merge mark, and c group fluorescent mark picture is the merging stack of a group fluorescent mark picture and b group fluorescent mark picture, and g group fluorescent mark picture is the merging stack of e group fluorescent mark picture and f group fluorescent mark picture.
(3) d, h group is amplified (Zoom) processing for do respectively image electronic on the basis of c, g group.
Fig. 6 B is that immunofluorescence detects the effect schematic diagram of described siRNA to bird flu H9N2 viruses adsorption A549 cell surface.In Fig. 6 B, left side one classifies the restraining effect schematic diagram of the non-targeted siRNA of transfection to bird flu H9N2 viruses adsorption A549 cell surface as, and the restraining effect schematic diagram of a siRNA cell of classifying transfection target ST3GAL4 gene as to bird flu H9N2 viruses adsorption A549 cell surface on right side.
Under laser confocal microscope, observe: a large amount of virion absorption is seen on bird flu H9N2 viruses adsorption A549 cell (left side) surface of the non-targeted siRNA of transfection; And the seldom virion absorption of amount is only seen on the bird flu H9N2 viruses adsorption A549 cell (right side) of the siRNA of transfection target ST3GAL4 gene surface.
The result being shown from Fig. 6 B, the siRNA of target ST3GAL4 gene has obvious restraining effect to bird flu H9N2 viruses adsorption A549 cell surface.Compare without target-spot siRNA with non-transfected cells and transfection, the virion of the siRNA treatment group cell surface absorption of target ST3GAL4 gene obviously reduces.
In Fig. 6 B, respectively organize accompanying drawing and adopt following mark mode: a, Anti-HA, b, DAPI, c, Merge, d, Zoom; E, Anti-HA, f, DAPI, g, Merge, h, Zoom, its mark mode is identical with Fig. 6 A, does not repeat them here.
Fig. 6 C is that immunofluorescence detects the effect schematic diagram of described siRNA to new first stream H1N1 influenza virus absorption HBE cell surface.In Fig. 6 C, one of left side classifies the restraining effect schematic diagram of the non-targeted siRNA of transfection to new first stream H1N1 influenza virus absorption HBE cell surface as, and a siRNA cell of classifying transfection target ST6GAL1 gene as on right side adsorbs the restraining effect schematic diagram of HBE cell surface to new first stream H1N1 influenza virus.
Under laser confocal microscope, observe: a large amount of virion absorption is seen on new first stream H1N1 influenza virus absorption HBE cell (left side) surface of the non-targeted siRNA of transfection; And the seldom virion absorption of amount is only seen on new first stream H1N1 influenza virus absorption HBE cell (right side) surface of the siRNA of transfection target ST6GAL1 gene.
The result being shown from Fig. 6 C, the siRNA of target ST6GAL1 gene has obvious restraining effect to new first stream H1N1 influenza virus absorption HBE cell surface.Compare without target-spot siRNA with non-transfected cells and transfection, the virion of the siRNA treatment group cell surface absorption of target ST6GAL1 gene obviously reduces.
In Fig. 6 C, respectively organize accompanying drawing and adopt following mark mode: a, Anti-HA, b, DAPI, c, Merge; D, Anti-HA, e, DAPI, f, Merge, its mark mode, with described in Fig. 6 A, does not repeat them here.
Fig. 6 D is that immunofluorescence detects the effect schematic diagram of described siRNA to bird flu H9N2 viruses adsorption HBE cell surface.In Fig. 6 D, left side one classifies the restraining effect schematic diagram of the non-targeted siRNA of transfection to bird flu H9N2 viruses adsorption HBE cell surface as, and the restraining effect schematic diagram of a siRNA cell of classifying transfection target ST3GAL4 gene as to bird flu H9N2 viruses adsorption HBE cell surface on right side.
Under laser confocal microscope, observe: a large amount of virion absorption is seen on bird flu H9N2 viruses adsorption HBE cell (left side) surface of the non-targeted siRNA of transfection; And the seldom virion absorption of amount is only seen on the bird flu H9N2 viruses adsorption HBE cell (right side) of the siRNA of transfection target ST3GAL4 gene surface.
The result being shown from Fig. 6 D, the siRNA of target ST3GAL4 gene has obvious restraining effect to bird flu H9N2 viruses adsorption HBE cell surface.Compare without target-spot siRNA with non-transfected cells and transfection, the virion of the siRNA treatment group cell surface absorption of target ST3GAL4 gene obviously reduces.
In Fig. 6 D, respectively organize accompanying drawing and adopt following mark mode: a, Anti-HA, b, DAPI, c, Merge; D, Anti-HA, e, DAPI, f, Merge, its mark mode, with described in Fig. 6 A, does not repeat them here.
Fig. 6 E is that immunofluorescence detects the effect schematic diagram of described siRNA to new first stream H1N1 influenza virus absorption HEp-2 cell surface.In Fig. 6 E, one of left side classifies the restraining effect schematic diagram of the non-targeted siRNA of transfection to new first stream H1N1 influenza virus absorption HEp-2 cell surface as, and a siRNA cell of classifying transfection target ST6GAL1 gene as on right side adsorbs the restraining effect schematic diagram of HEp-2 cell surface to new first stream H1N1 influenza virus.
Under laser confocal microscope, observe: a large amount of virion absorption is seen on new first stream H1N1 influenza virus absorption HEp-2 cell (left side) surface of the non-targeted siRNA of transfection; And the seldom virion absorption of amount is only seen on new first stream H1N1 influenza virus absorption HEp-2 cell (right side) surface of the siRNA of transfection target ST6GAL1 gene.
The result being shown from Fig. 6 E, the siRNA of target ST6GAL1 gene has obvious restraining effect to new first stream H1N1 influenza virus absorption HEp-2 cell surface.Compare without target-spot siRNA with non-transfected cells and transfection, the virion of the siRNA treatment group cell surface absorption of target ST6GAL1 gene obviously reduces.
In Fig. 6 E, respectively organize accompanying drawing and adopt following mark mode: a, Anti-HA, b, DAPI, c, Merge; D, Anti-HA, e, DAPI, f, Merge, its mark mode, with described in Fig. 6 A, does not repeat them here.
Fig. 6 F is that immunofluorescence detects the effect schematic diagram of described siRNA to bird flu H9N2 viruses adsorption HEp-2 cell surface.In Fig. 6 F, left side one classifies the restraining effect schematic diagram of the non-targeted siRNA of transfection to bird flu H9N2 viruses adsorption HEp-2 cell surface as, and the restraining effect schematic diagram of a siRNA cell of classifying transfection target ST3GAL4 gene as to bird flu H9N2 viruses adsorption HEp-2 cell surface on right side.
Under laser confocal microscope, observe: a large amount of virion absorption is seen on bird flu H9N2 viruses adsorption HEp-2 cell (left side) surface of the non-targeted siRNA of transfection; And the seldom virion absorption of amount is only seen on the bird flu H9N2 viruses adsorption HEp-2 cell (right side) of the siRNA of transfection target ST3GAL4 gene surface.
The result being shown from Fig. 6 F, the siRNA of target ST3GAL4 gene has obvious restraining effect to bird flu H9N2 viruses adsorption HEp-2 cell surface.Compare without target-spot siRNA with non-transfected cells and transfection, the virion of the siRNA treatment group cell surface absorption of target ST3GAL4 gene obviously reduces.
In Fig. 6 F, respectively organize accompanying drawing and adopt following mark mode: a, Anti-HA, b, DAPI, c, Merge; D, Anti-HA, e, DAPI, f, Merge, its mark mode, with described in Fig. 6 A, does not repeat them here.
In sum, the siRNA treatment group of target ST6GAL1 and ST3GAL4 gene has obvious restraining effect to the surface of airway epithelial cell system (A549, HBE and HEp-2 cell), and the virion of described cell surface absorption obviously reduces.
Embodiment six, infect described siRNA infected by influenza in 4 hours and enter the impact in transfectional cell
Described siRNA is imported to airway epithelial cell, and method is with embodiment bis-, transfection after 72 hours, join in the stream of people Influenza Virus H1N1 and avian influenza virus H9N2, and with non-transfected cells and transfection without target-spot siRNA in contrast.In virus inoculation 4 hours, quantitative RT-PCR detected result shows: the intracellular virogene quantity of described siRNA treatment group obviously reduces (as shown in Figure 7 A, 7 B).
Fig. 7 A is that quantitative RT-PCR detect to infect the schematic diagram that described siRNA in 4 hours enters the human influenza virus H1N1 gene dosage in transfectional cell.Fig. 7 A has shown with non-transfected cells and transfection and has compared without target-spot siRNA, the intracellular virogene quantity of A549 of the siRNA treatment group of transfection si-ST6GAL1-001 and si-ST6GAL1-002 obviously reduces, and its human influenza virus H1N1 gene dosage has roughly reduced 70 ~ 80%; The intracellular virogene quantity of HBE of the siRNA treatment group of transfection si-ST6GAL1-001 and si-ST6GAL1-002 obviously reduces, and its human influenza virus H1N1 gene dosage has roughly reduced 70 ~ 80%; The intracellular virogene quantity of HEp-2 of the siRNA treatment group of transfection si-ST6GAL1-001 and si-ST6GAL1-002 obviously reduces, and its human influenza virus H1N1 gene dosage has roughly reduced 70 ~ 80%.
The result being shown from Fig. 7 A, the siRNA of target ST6GAL1 gene can obviously reduce new first stream H1N1 virus and enter airway epithelial cell.
It should be noted that, the siRNA treatment group of transfection si-ST6GAL1-001, si-ST6GAL1-002 in Fig. 7 A and transfection have significant difference ， ﹡ without the intracellular H1N1 gene dosage of target-spot siRNA treatment group
p< 0.05.
Fig. 7 B is that quantitative RT-PCR detect to infect the schematic diagram that described siRNA in 4 hours enters the avian influenza virus H9N2 gene dosage in transfectional cell.From Fig. 7 B, compare without target-spot siRNA with non-transfected cells and transfection, the intracellular virogene quantity of A549 of the siRNA treatment group of transfection si-ST3GAL4-001 and si-ST3GAL4-002 obviously reduces, and its avian influenza virus H9N2 gene dosage has roughly reduced 70 ~ 80%; The intracellular virogene quantity of HBE of the siRNA treatment group of transfection si-ST3GAL4-001 and si-ST3GAL4-002 obviously reduces, and its avian influenza virus H9N2 gene dosage has roughly reduced 70 ~ 80%; The intracellular virogene quantity of HEp-2 of the siRNA treatment group of transfection si-ST3GAL4-001 and si-ST3GAL4-002 obviously reduces, and its avian influenza virus H9N2 gene dosage has roughly reduced 70 ~ 80%.
The result being shown from Fig. 7 B, the siRNA of target ST3GAL4 gene can obviously reduce bird flu H9N2 cell entry airway epithelial cell.
It should be noted that, the transfection si-ST3GAL4-001 in Fig. 7 B, the siRNA treatment group of si-ST3GAL4-002 and transfection have significant difference ， ﹡ without the intracellular H9N2 gene dosage of target-spot siRNA treatment group
p< 0.05.
In sum, the intracellular H1N1 of siRNA treatment group and the H9N2 virogene quantity of target ST6GAL1 and ST3GAL4 gene obviously reduce, and the siRNA of target ST6GAL1 and ST3GAL4 gene can obviously reduce initial infection H1N1 and H9N2 cell entry airway epithelial cell.
Embodiment seven, the many replicative cycles of described siRNA infected by influenza infect the restraining effect of airway epithelial
Described siRNA is imported to airway epithelial cell, and method is with embodiment bis-, transfection after 72 hours, join in the stream of people Influenza Virus H1N1 or avian influenza virus H9N2, and with non-transfected cells and transfection without target-spot siRNA in contrast.Respectively at different time points after infecting, collecting cell culture supernatant is made TCID50(Tissue culture infective dose) test.Result shows, after described siRNA interference goal gene, can effectively suppress infect (as Fig. 8 A, Fig. 8 B as shown in) of virus to airway epithelial cell.
Fig. 8 A is that described siRNA infects the restraining effect schematic diagram of airway epithelial to the many replicative cycles of human influenza virus H1N1.Fig. 8 A has shown siRNA treatment group cell measured H1N1 virus titre (Log TCID of different time points after infecting H1N1 of transfection si-ST6GAL1
50), as seen from the figure, behind 36 hours of infection H1N1, H1N1 virus titre (the Log TCID of the siRNA treatment group cell of transfection si-ST6GAL1
50) obviously low without target-spot siRNA treatment group compared with non-transfected cells and transfection.
The result being shown from Fig. 8 A, the siRNA of target ST6GAL1 gene has long-time restraining effect to new first stream H1N1 virus.
It should be noted that, the siRNA treatment group of the transfection si-ST6GAL1 in Fig. 8 A and transfection have significant difference ， ﹡ without the H1N1 virus titre of target-spot siRNA treatment group cell
p< 0.05.
Fig. 8 B is that described siRNA infects the restraining effect schematic diagram of airway epithelial to the many replicative cycles of bird flu H9N2.Fig. 8 B has shown siRNA treatment group cell measured H9N2 virus titer (Log TCID of different time points after infecting H9N2 of transfection si-ST3GAL4
50), as seen from the figure, behind 36 hours of infection H9N2, the H9N2 virus titer (Log TCID50) of the siRNA treatment group cell of transfection si-ST3GAL4 is obviously low without target-spot siRNA treatment group compared with non-transfected cells and transfection.
The result being shown from Fig. 8 B, the siRNA of target ST3GAL4 gene has long-acting restraining effect to bird flu H9N2 virus.
It should be noted that, the siRNA treatment group of the transfection si-ST3GAL4 in Fig. 8 B and transfection have significant difference ， ﹡ without the H9N2 virus titer of target-spot siRNA treatment group cell
p< 0.05.
In sum, the treatment group of target ST6GAL1 and ST3GAL4 gene can suppress H1N1 and H9N2 virus infecting airway epithelial cell for a long time, effectively.
The pharmaceutical composition of embodiment eight, described siRNA sequence suppresses the effect of multiple influenza virus sub-strain
Specific siRNA drug combination (siST6GAL1-001+siST3GAL4-001, siST6GAL1-001+siST3GAL4-002, siST6GAL1-002+siST3GAL4-001, siST6GAL1-002+siST3GAL4-002) is turned to airway epithelial cell with 5nM concentration, transfection method is with embodiment bis-, and detection method is with embodiment five, six.Result demonstration, Combined Preparation group siRNA all reaches obvious inhibition (as shown in Fig. 9 A and Fig. 9 B) to people source and fowl source and course Influenza Virus.
Fig. 9 A is the pharmaceutical composition of the described siRNA sequence restraining effect schematic diagram to human influenza virus H1N1 Subtypes.Fig. 9 A has shown: compares without target-spot siRNA with non-transfected cells and transfection,
(1) the intracellular human influenza virus's H1N1 virus of A549 titre (the Log TCID of si-ST6GAL1-001+ si-ST3GAL4-001 pharmaceutical composition, si-ST6GAL1-001+ si-ST3GAL4-002 pharmaceutical composition, si-ST6GAL1-002+ si-ST3GAL4-001 pharmaceutical composition, si-ST6GAL1-002+ si-ST3GAL4-002 pharmaceutical composition treatment group
50) obviously reduce;
(2) the intracellular human influenza virus's H1N1 virus of HBE titre (the Log TCID of si-ST6GAL1-001+ si-ST3GAL4-001 pharmaceutical composition, si-ST6GAL1-001+ si-ST3GAL4-002 pharmaceutical composition, si-ST6GAL1-002+ si-ST3GAL4-001 pharmaceutical composition, si-ST6GAL1-002+ si-ST3GAL4-002 pharmaceutical composition treatment group
50) obviously reduce;
(3) the intracellular human influenza virus's H1N1 virus of HEp-2 titre (the Log TCID of si-ST6GAL1-001+ si-ST3GAL4-001 pharmaceutical composition, si-ST6GAL1-001+ si-ST3GAL4-002 pharmaceutical composition, si-ST6GAL1-002+ si-ST3GAL4-001 pharmaceutical composition, si-ST6GAL1-002+ si-ST3GAL4-002 pharmaceutical composition treatment group
50) obviously reduce;
The result being shown from Fig. 9 A, the siRNA combination of target ST6GAL1 and ST3GAL4 has obvious restraining effect to new first stream H1N1 virus Subtypes.
It should be noted that, the pharmaceutical composition treatment group of the siRNA sequence in Fig. 9 A and transfection have significant difference ， ﹡ without the H1N1 virus titre of target-spot siRNA treatment group cell
p< 0.05.
Fig. 9 B is the pharmaceutical composition of the described siRNA sequence restraining effect schematic diagram to bird flu H9N2 Subtypes.Fig. 9 B has shown: compares without target-spot siRNA with non-transfected cells and transfection,
(1) the intracellular bird flu H9N2 of A549 virus titer (the Log TCID of si-ST6GAL1-001+ si-ST3GAL4-001 pharmaceutical composition, si-ST6GAL1-001+ si-ST3GAL4-002 pharmaceutical composition, si-ST6GAL1-002+ si-ST3GAL4-001 pharmaceutical composition, si-ST6GAL1-002+ si-ST3GAL4-002 pharmaceutical composition treatment group
50) obviously reduce;
(2) the intracellular bird flu H9N2 of HBE virus titer (the Log TCID of si-ST6GAL1-001+ si-ST3GAL4-001 pharmaceutical composition, si-ST6GAL1-001+ si-ST3GAL4-002 pharmaceutical composition, si-ST6GAL1-002+ si-ST3GAL4-001 pharmaceutical composition, si-ST6GAL1-002+ si-ST3GAL4-002 pharmaceutical composition treatment group
50) obviously reduce;
(3) the intracellular bird flu H9N2 of HEp-2 virus titer (the Log TCID of si-ST6GAL1-001+ si-ST3GAL4-001 pharmaceutical composition, si-ST6GAL1-001+ si-ST3GAL4-002 pharmaceutical composition, si-ST6GAL1-002+ si-ST3GAL4-001 pharmaceutical composition, si-ST6GAL1-002+ si-ST3GAL4-002 pharmaceutical composition treatment group
50) obviously reduce;
The result being shown from Fig. 9 B, the siRNA composition of target ST6GAL1 and ST3GAL4 infects and has obvious restraining effect bird flu H9N2 virus subtype.
It should be noted that, the pharmaceutical composition treatment group of the siRNA sequence in Fig. 9 B and transfection have significant difference ， ﹡ without the H9N2 virus titer of target-spot siRNA treatment group cell
p< 0.05.
In sum, target ST6GAL1 and ST3GAL4 gene siRNA combination H1N1 and H9N2 virus subtype are infected and all reach obvious inhibition.
Embodiment nine, described siRNA sequence produce the impact of interferon-' alpha ' on transfered cell
Consider part siRNA can cause Cells Interferon Production-α (INF-α) and play non-specific antivirus action, therefore apply ELISA method (enzyme-linked immunosorbent assay, enzyme-linked immunosorbent assay) detect, transfection method is with embodiment bis-, proceed to after 48 hours, collecting cell supernatant detects (as shown in figure 10).Result demonstration, described siRNA does not all cause the generation of nonspecific INF-α.
The above is the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications are also considered as protection scope of the present invention.
Claims (9)
1. can prevent and treat a siRNA for influenza, it is characterized in that, described siRNA be following at least one:
SiST6GAL1_001: positive-sense strand based composition is SEQ ID NO.1, antisense strand based composition is SEQ ID NO.2;
SiST6GAL1_002: positive-sense strand based composition is SEQ ID NO.3, antisense strand based composition is SEQ ID NO.4;
SiST3GAL4_001: positive-sense strand based composition is SEQ ID NO.5, antisense strand based composition is SEQ ID NO.6;
SiST3GAL4_002: positive-sense strand based composition is SEQ ID NO.7, antisense strand based composition is SEQ ID NO.8.
2. siRNA according to claim 1, is characterized in that, 3 ' the terminal modified bases of dangling of every chain of described siRNA.
3. siRNA according to claim 2, is characterized in that, described in the base one or more deoxyribonucleotides in dA, dT, dG, dC that dangle form.
4. siRNA according to claim 2, is characterized in that, described in base any two deoxyribonucleotides in dA, dT, dG, dC that dangle form.
5. siRNA according to claim 1, is characterized in that, described siRNA is through the one or more modification in phosphorylation, methoxylation, sulfo-, cholesterol, Cy3, Cy5, vitamin H, VITAMIN, folic acid, cholic acid, PEG.
6. siRNA according to claim 1, is characterized in that, described siRNA also process is the modification of 2 '-O-methylribonucleotide and/or 2 '-fluoro.
7. according to the siRNA described in claim 1-6 any one, it is characterized in that, described siRNA is the mixing of two kinds of siST6GAL1_001 and siST3GAL4_001, the mixing that siST6GAL1_001 and siST3GAL4_002 are two kinds, the mixing that siST6GAL1_002 and siST3GAL4_001 are two kinds, or the mixing of two kinds of siST6GAL1_002 and siST3GAL4_002.
8. can prevent and treat a pharmaceutical composition for influenza, it is characterized in that, its active ingredient is the siRNA described in claim 1-7 any one.
9. the medicinal use of the siRNA described in claim 1 ~ 7 any one, is characterized in that, for the preparation of pharmaceutical composition, medicine and the preventing preparation that can prevent and treat influenza virus.
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| RU2609857C1 (en) * | 2015-11-02 | 2017-02-06 | Федеральное государственное бюджетное учреждение "Государственный научный центр "Институт иммунологии" Федерального медико-биологического агентства России (ФГБУ "ГНЦ Институт иммунологии" ФМБА России) | Application of composition consisting of cationic ltp peptide and molecules of rna against respiratory viruses |
| CN108182346A (en) * | 2016-12-08 | 2018-06-19 | 杭州康万达医药科技有限公司 | Predict method for building up and its application of the siRNA for the machine learning model of the toxicity of certain class cell |
| WO2022012663A1 (en) * | 2020-07-17 | 2022-01-20 | 创观（苏州）生物科技有限公司 | Virus insusceptible animal and construction method therefor |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| RU2609857C1 (en) * | 2015-11-02 | 2017-02-06 | Федеральное государственное бюджетное учреждение "Государственный научный центр "Институт иммунологии" Федерального медико-биологического агентства России (ФГБУ "ГНЦ Институт иммунологии" ФМБА России) | Application of composition consisting of cationic ltp peptide and molecules of rna against respiratory viruses |
| CN108182346A (en) * | 2016-12-08 | 2018-06-19 | 杭州康万达医药科技有限公司 | Predict method for building up and its application of the siRNA for the machine learning model of the toxicity of certain class cell |
| CN108182346B (en) * | 2016-12-08 | 2021-07-30 | 杭州康万达医药科技有限公司 | Establishment method and application of machine learning model for predicting toxicity of siRNA to certain cells |
| WO2022012663A1 (en) * | 2020-07-17 | 2022-01-20 | 创观（苏州）生物科技有限公司 | Virus insusceptible animal and construction method therefor |
| WO2022199690A1 (en) * | 2021-03-26 | 2022-09-29 | 圣诺生物医药技术(苏州)有限公司 | Sirna drug, pharmaceutical composition, sirna-small molecule drug conjugate, and application thereof |
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