CN104673796A - Small-interference RNA of target HSV-1 virus UL18 gene and application of small-interference RNA - Google Patents

Abstract

The invention discloses a small-interference RNA of a target HSV-1 virus UL18 gene and application of the small-interference RNA. The small-interference RNA is designed and synthesized according to an mRNA sequence of the target HSV-1 virus UL18 gene. The invention firstly finds that the proliferation and infection of HSV-1 are finally inhibited because siRNA of the UL18 gene can cause the condition that an HSV-1 nucleocapsid cannot be normally assembled; and meanwhile the invention finds that the siRNA, serving as a small-molecular nucleotide drug, of the target HSV-1 virus UL18 gene, also has the effect of inhibiting a drug-resistant strain of acyclovir in vitro. By using the small-interference RNA, the problems that the drug toxicity is high, the drug resistance is easily generated, diseases easily recur and the like in the existing HSV-1 infection treatment scheme can be solved; the siRNA and modified substances thereof can be used for preparing the small-molecular nucleotide drug or preparation for preventing or treating HSV-1 and HSV-1 virus infection related diseases; and the small-interference RNA is clear in targeting and remarkable in effect.

Description

The siRNA of target HSV-1 virus UL18 gene and application

Technical field

The invention belongs to field of molecular biotechnology, particularly the siRNA molecule (siRNA) of target HSV-1 virus UL18 gene and application.

Background technology

Hsv (Herpes simplex virus, HSV) belongs to herpetoviridae, Alphaherpesvirinae, and the double-strand coating DNA virus of unique natural reservoir (of bird flu viruses) that to be a kind of with the mankind be, serology is divided into HSV-1 and HSV-2 type.HSV-1, after the bleb disease damage forming local, usually causes the infection of organism nervous system in every way further.The particularly important is in the neural process of infection, simplexvirus can play mode the most stunning in its existence skill :--reactivation-this biological superiority formed in evolution of hiding, make HSV-1 can avoid the attack of host immune system to a great extent, thus stop even throughout one's life for a long time in body.The normal recurrent exerbation of HSV-1 infectious diseases, delay is difficult, causes considerable distress to patient.At present, pharmacological agent is the Main Means that treatment HSV infects.Clinical conventional medicine is ucleosides and the analogue thereof such as acycloguanosine mainly, as acyclovir (Acyclovir, ACV), valacyclovir etc.Its action target spot is viral dna polymerase, suppresses copying of virus.Heparin can with viral membrane surface Glycoprotein binding; Compound S CH 43478 and analogue thereof suppress the expression of the immediate early gene of HSV; Thiourea inhibitors WAY-150138 can suppress UL6 to participate in Mouth Disease Virus Proteins, suppresses progeny viral DNA packaging to enter capsid structure.But partial nucleotide analogue such as idoxene, ganciclovir etc. have mutagenicity, and security is low, and the eighties in last century ACV persister namely occur, and widely using along with anti-herpesvirus medicament, the report about persister also gets more and more.So, find the R&D target that the methods for the treatment of of new anti-herpesvirus and therapy target become researcher.

RNA interference (RNA interference, RNAi) is the expression of specific gene silencing phenomenon that double-stranded RNA (double-stranded RNA, dsRNA) mediates.In cell, dsRNA is cut into short dsRNA (21 ~ 25nt) by rnase (also known as Dicer), and this little dsRNA is referred to as siRNA.Antisense strand in siRNA and multiple nucleic acids enzyme form complex body and are called RISC complex body.RISC complex body is activated subsequently becomes active condition (RISC*).The positive-sense strand of siRNA guides RISC* to reticent target spot, RISC* is combined with reticent target spot mRNA, under the existence of ATP, the antisense strand of mRNA and siRNA combines complementary, cause nuclease and mRNA is sheared the small segment becoming 21 ~ 23nt, then stop the expression of mRNA to be translated, cause the silence of goal gene.There are some researches show that siRNA has great development prospect as efficiently special antiviral, siRNA is at HIV-1, and SARS virus, is successfully applied in the Therapy study of the multiple viruses such as respiratory complication virus.

Summary of the invention

In order to overcome the shortcoming of prior art with not enough, primary and foremost purpose of the present invention is the siRNA molecule (siRNA) and the modifier thereof that provide target HSV-1 virus UL18 gene.Solve the drug toxicity existed in existing HSV-1 treatment of infection scheme and produce resistance greatly, easily, defect and the problems such as easy recurrence, obtained target is clear and definite, safe and reliable, the Novel medicine of the anti-HSV-1 virus infection of successful.

Another object of the present invention is to the application of the siRNA that target HSV-1 virus UL18 gene is provided.

Object of the present invention is achieved through the following technical solutions: the siRNA of target HSV-1 virus UL18 gene, described siRNA are one in siRNA181, siRNA182 or siRNA183 or at least two kinds:

SiRNA181: positive-sense strand: 5'-CUCAACGUGCUGUACUACAAC-3';

Antisense strand: 5'-UGUAGUACAGCACGUUGAGGU-3';

SiRNA182: positive-sense strand: 5'-CCACCAUCAUCCUUACGCUAA-3';

Antisense strand: 5'-AGCGUAAGGAUGAUGGUGGUU-3';

SiRNA183: positive-sense strand: 5'-CCCGUUAUACGCUAUCCCUAA-3';

Antisense strand: 5'-AGGGAUAGCGUAUAACGGGGG-3';

SiRNA N.C: positive-sense strand: 5'-UUCUCCGAACGUGUCACGUTT-3';

Antisense strand: 5'-ACGUGACACGUUCGGAGAATT-3';

Wherein, siRNA N.C is siRNA negative control, and the TT base that its 3' holds is overhanging sequence, is to increase siRNA in intracellular stability.

The siRNA molecule for HSV-1 virus UL18 gene in the present invention, can carry out according to actual needs modifying the modification mode primarily of following: (1) end modified mode (generally terminal modified at positive-sense strand 5'): fluorophor is modified: FAM, CY3 etc., modifying object is be convenient to observe; Specific groups is modified: vitamin H, amino, cholesterol, phosphate, S-S modify, and wherein cholesterol modifies object is be convenient to enter cell, and other modify object is utilize end group to carry out other experiments.(2) base modification mode: the modification of 2' methoxy, the modification of 2' fluoro, thio-modification etc., modifying object is increase stability.

Described siRNA and derivative modifier can be used for medicine or the preparation of preparing prevention or treatment HSV-1 and any disease relevant to HSV-1 virus infection.Topmost purposes comprises: make eye drop, can be used for prevention and therapy HSV-1 and infects the eye illness caused; As basting agent, can be used for prevention and therapy HSV-1 and infect the herpes labialis, the genital herpes that cause; As injection, can prevention and therapy HSV-1 latent infection and recurrence etc.

The present invention, relative to prior art, has following advantage and effect:

(1) the present invention overcome existing treatment HSV-1 infect drug toxicity produce resistance greatly, easily, the defects such as easy recurrence and problem, and obtain all siRNA molecules of anti-HSV-1 (siRNA) product for UL18 gene and modifier thereof by certain technological means.

(2) siRNA molecule (siRNA) for the treatment of provided by the invention or prevention HSV-1 virus infection, target is clear and definite, successful;

(3) provided by the present invention treated by siRNA or prevent the novel method of HSV-1 virus infection be simplexvirus control a kind of new approaches.

Accompanying drawing explanation

Fig. 1 is the cytotoxicity result figure of different concns siRNA and lipofectamineRNAiMAX.

Fig. 2 is the result figure that the agarose gel electrophoresis of total serum IgE in embodiment 4 detects.

Fig. 3 is the result figure of Real time PCR melting curve; Wherein, Fig. 3 A is the melting curve of internal reference primer GAPDH and UL18 gene test primer; Fig. 3 B is the melting peak of internal reference primer GAPDH and UL18 gene test primer.

Fig. 4 is that Real-time PCR detects the inhibiting result figure of siRNA interference to UL18 gene mRNA.

Fig. 5 is the result figure of flow cytomery UL18-EGFP fusion rotein fluorescence relative expression intensities.

Fig. 6 is the result figure that siRNA disturbs the impact of UL18 gene pairs HSV-1 plaque test; Wherein, Fig. 6 A is the plaque that different experiments group is formed; Fig. 6 B is the plaque test amount of each experimental group after three kinds of siRNA interference.

Fig. 7 is the result figure of the impact of born of the same parents' inner virus number of particles after siRNA interference UL18 gene pairs HSV-1 cells infected; Wherein, Fig. 7 A is that HSV-1 infects control group; Fig. 7 B is negative control siN.C control group; Fig. 7 C is siRNA182 interference group.

Embodiment

Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited thereto.

The experimental technique of unreceipted specific experiment condition in the following example, usually conveniently experiment condition or the experiment condition of advising according to manufacturer.

Embodiment 1

The present invention is directed to HSV-1 virus UL18 gene nucleotide series, design siRNA double-strand sequence, close and suppress the expression of HSV-1UL18 gene, the nucleocapsid of HSV-1 is caused normally to assemble, thus the final HSV-1 of suppression breeds and infects, resistance is produced greatly, easily, defect and the problems such as easy recurrence to solve the drug toxicity existed in existing HSV-1 treatment of infection scheme; SiRNA double-strand sequence provided by the present invention can be used for medicine or the preparation of preparing prevention or treatment HSV-1 and any disease relevant to HSV-1 virus infection.When this siRNA double-strand sequence is entered in organism by certain mode, the expression of suppression HSV-1 disease-related capsid protein that can be efficient, special, related gene is occurred reticent, thus the expression of target gene is effectively knocked out, reach treatment and the object of preventing HSV-1 and the disease relevant to HSV-1 virus infection.Target of the present invention is clear and definite, Be very effective.

The present invention, according to HSV-1UL18 gene order (Genebank accession number: AB618031.1), submits the coding region sequence of UL18 gene to siRNA Photographing On-line website http://jura.wi.mit.edu/bioc/siRNA; Https: //www.genscript.com/ssl-bin/app/rnai; Http:// www.ambion.com/techlib/misc/siRNA-finder.html, according to the siRNA that siRNA principle of design primary design is potential with screening.Submit UL18 gene order to http://www.ncbi.nlm.nih.gov/BLAST website, the conservative property of analyzing gene sequence, choose the potential target position of the common conservative region between different strain and kind as siRNA, select the potential siRNA being positioned at conservative region as far as possible.Selected siRNA sequence is submitted to http://www.ncbi.nlm.nih.gov/BLAST web analytics homology, and analyze target sequence and designed siRNA with RNA secondary structure analysis software RNAdraw1.1 and RNAstructure 4.2, avoid the formation of stronger secondary structure.

The siRNA (siRNA181, siRNA182, siRNA183) of 3 pairs of target HSV-1UL18 genes is devised according to above principle, submit the siRNA designed the synthesis of to Shanghai Ji Ma company, synthesize siRNA negative control siRNAN.C (interference effect can not be had to any gene) simultaneously.

SiRNA and the siRNA negative control siRNA N.C of 3 pairs of target HSV-1 virus UL18 genes of design and synthesis, its sequence is as follows:

SiRNA181: positive-sense strand: 5'-CUCAACGUGCUGUACUACAAC-3';

Antisense strand: 5'-UGUAGUACAGCACGUUGAGGU-3';

SiRNA182: positive-sense strand: 5'-CCACCAUCAUCCUUACGCUAA-3';

Antisense strand: 5'-AGCGUAAGGAUGAUGGUGGUU-3';

SiRNA183: positive-sense strand: 5'-CCCGUUAUACGCUAUCCCUAA-3';

Antisense strand: 5'-AGGGAUAGCGUAUAACGGGGG-3';

SiRNA N.C: positive-sense strand: 5'-UUCUCCGAACGUGUCACGUTT-3';

Antisense strand: 5'-ACGUGACACGUUCGGAGAATT-3';

Wherein, the TT base of the 3' end in siRNA N.C is overhanging sequence, is to increase siRNA in intracellular stability.

The siRNA molecule for HSV-1 virus UL18 gene in the present invention, can carry out according to actual needs modifying the modification mode primarily of following: (1) end modified mode (generally terminal modified at positive-sense strand 5'): fluorophor is modified: FAM, CY3 etc., modifying object is be convenient to observe; Specific groups is modified: vitamin H, amino, cholesterol, phosphate, S-S modify, and wherein cholesterol modifies object is be convenient to enter cell, and other modify object is utilize end group to carry out other experiments.(2) base modification mode: the modification of 2' methoxy, the modification of 2' fluoro, thio-modification etc., modifying object is increase stability.

The cultivation of embodiment 2HSV-1 virus and titer determination

(1) cultivation of HSV-1 virus

Middle flask culture Vero cell (ATCC CCL81, purchased from ATCC company) grows to cell 50% and converges, and abandons nutrient solution, and PBS damping fluid (0.01M, pH 7.4) washes 1 ~ 2 time.Take out-80 DEG C and preserve HSV-1F strain virus (purchased from Wuhan Virology Institute,Chinan academy of Sciences), melt rapidly, the viral suspension of 200 μ L is added culturing bottle, in 37 DEG C, 5% (v/v) CO ₂adsorb 2h in incubator, period is every the slow shaken several times of 15min.Add cell culture medium DMEM (purchased from life company) maintenance medium 4mL, put 37 DEG C, 5%CO ₂incubator continues to cultivate.After the complete pathology of basis of microscopic observation cell, by cell freeze thawing three times, cryopreservation tube packing virus-culturing fluid ,-80 DEG C save backup.

(2) TCID ₅₀measure

Vero cell adjustment cell count 1.5 × 10 ⁵cells/mL adds 96 porocyte culture plates and cultivates, 100 μ L/ holes, and 37 DEG C, 5%CO2 incubator overnight incubation, treats that cell grows up to individual layer, by the TCID of Endpoint Dilution Method titration virus ₅₀.By maintenance medium 10 times of serial dilution viruses.Inoculation serial dilution virus liquid, each extent of dilution establishes 8 multiple holes, establishes normal cell controls hole simultaneously.96 orifice plates are placed in 37 DEG C, 5%CO ₂cultivate, observe day by day and record occur cytopathic hole count, to cytopathy not developing deeply time, by Reed-Muench method calculating TCID ₅₀.

TCID ₅₀=pathology rate higher than 50% group of dilution logarithmic value+distance than.

Result shows: the 50tissue infection dose (TCID of HSV-1 ₅₀) be 10 ^-7.5/ 100 μ L, namely 100 μ L titres are inoculated in every hole is 10 ^-7.5hSV-1, can make the cell generation pathology of 50%.

Embodiment 3siRNA N.C and transfection reagent cytotoxic assay

Mtt assay mensuration siRNA N.C and transfection reagent are to the toxicity of Vero cell: 1. cell is after trysinization, adds in 96 well culture plates, and 100 μ L/ holes, cell density reaches 1.5 × 10 ⁵nutrient solution is abandoned after cells/mL, 24h.2. respectively by different concns (pmol/cm ²) siRNA N.C be diluted in 25 μ L OPTI-MEM transfections nutrient solution (purchased from life company), mixing; 3. separately get different concns transfection reagent LipofectamineRNAiMAX (purchased from Invitrogen company) and be diluted in 25 μ L OPTI-MEM transfection nutrient solutions, incubated at room 5min; 4. 2. step is mixed with step transfection nutrient solution 3., form transfection cocktail, incubated at room 20min; Establish 16 different concns dosage groups altogether; 5. by 96 good in advance porocyte culture plates, inhale and abandon culture supernatant, every hole adds 50 μ L transfection cocktail, and vibration plate mixing gently, in 37 DEG C, 5%CO ₂incubator continues to cultivate; 6. after transfection 4h, inhale and abandon transfection cocktail, add cell growth medium (DMEM substratum contains 10% inactivated fetal bovine serum, purchased from life company), 96 orifice plates are placed in 37 DEG C, 5%CO ₂48h cultivated by incubator.7. every hole adds 5mg/mL MTT 10 μ L, and continue to cultivate 4h, after abandoning MTT liquid, every hole adds DMSO (dimethyl sulfoxide (DMSO)) 100 μ L, continues to cultivate 1h.8. elisa reading instrument colorimetric (wavelength 570nm, reference wavelength 630nm), surveys absorbance A value.Cell survival rate (%)=experimental group A570/ control group A 570 × 100% (see Fig. 1).

Experimental result shows: siRNA N.C and transfection reagent all have no significant effect cell survival rate, in table 1 under each dosage conditions.

Table 1 different concns siRNA N.C and lipofectamineRNAiMAX cytotoxicity (x ± s, n=4)

Embodiment 4siRNA is to the effect of HSV-1 virus

1, HSV-1 virus mRNA level detection in cell

(1) cell sample preparation:

(1) the Vero cell dilution of trysinization is 5.0 × 10 by Vero cell growth medium (DMEM substratum is containing 10% inactivated fetal bovine serum) ⁵cells/mL, 2mL/ implant in hole 6 porocyte culture plates, are placed in 37 DEG C, 5%CO ₂incubator is cultured to cell 90% and converges;

(2) getting 100pmol siRNA (see table 2) is diluted in 250 μ L OPTI-MEM transfection nutrient solutions, mixing;

(3) get 5 μ L Lipofectamine RNAiMAX and be diluted in 250 μ L OPTI-MEM transfection nutrient solutions, incubated at room 5min;

(4) the transfection nutrient solution of step (2) with step (3) is mixed, form transfection cocktail, incubated at room 20min;

(5) 6 orifice plate culture supernatant are abandoned in suction, and every hole adds 1000 μ L OPTI-MEM transfection nutrient solutions and 500 μ L transfection cocktail, and vibration plate mixing gently, in 37 DEG C, 5%CO ₂incubator continues to cultivate;

(6), after transfection 4h, inhale and abandon transfection cocktail, add HSV-1 viral dilution liquid (MOI=1) 100 μ L, 37 DEG C, 5%CO ₂incubator continues to cultivate, and every 15min gently rocker once, makes virus fully cells infected;

(7), after virus infection 2h, inhale and abandon virus liquid, add 2mL maintain liquid DMEM, 37 DEG C, 5%CO ₂incubator continues to cultivate; Collecting cell extracted total RNA after 48h.

The siRNA sequence of table 2 target UL18 gene

(2) cell total rna extracts:

(1) Trizol reagent (purchased from Invitrogen company), cracking sick cell: abandon nutrient solution, PBS washes one time, by 1mL Trizol/10cm ²the ratio of cell adds 1mL Trizol, and room temperature places 5min, makes the abundant cracking of cell.

(2) blow and beat cell, 1.5mL EP lysate being transferred to DEPC process manages.

(3) add 200 μ L chloroforms in the ratio of 200 μ L chloroform/mL Trizol, up and down violent upset mixing 15s, room temperature places 2 ~ 3min.

(4) 4 DEG C, be no more than 12000g (11970g) centrifugal 15min.

(5) slight absorption upper strata aqueous phase is to another centrifuge tube.

(6) add 500 μ L Virahols in the ratio of 0.5mL Virahol/mL Trizol, mixing, room temperature places 5 ~ 10min.

(7) 4 DEG C are no more than 12000g (11970g) centrifugal 10min, and inhale and abandon supernatant, RNA is sunken at the bottom of pipe.

(8) add 1000 μ L 75% (v/v) ethanol (-20 DEG C of precoolings) in the ratio of 1mL 75% ethanol/mL Trizol, carefully blow and beat precipitation.

(9) 4 DEG C are no more than 7500g (7490g) centrifugal 5min, abandon supernatant as far as possible.

(10) drying at room temperature 5 ~ 10min (too not dry, to precipitate into gel).

(11) add 50 μ L DEPC water, carefully blow and beat sample dissolution.-80 DEG C of preservations.

(12) get 5 μ L samples, dilute 20 times, nucleic acid-protein analyser is determined RNA purity and content.

After siRNA transfection is entered cell, HSV-1 infects, after HSV-1 infects 24h, and extracting RNA.Whether the RNA obtained carries out 1% agarose gel electrophoresis detection RNA with 3 μ L and degrades.Electrophoresis showed three signature bands, prove that the RNA extracted is not degraded (see Fig. 2), can be used for follow-up test.

(3) reverse transcription:

Following reagent is added in the PCR pipe of 0.2mL DEPC process:

Reaction conditions: 25 DEG C, 5min; 42 DEG C, 30min; 85 DEG C, 5min; 0 DEG C, forever.CDNA product-20 DEG C preservation.

(4) Real-time PCR:

△ △ Ct method detects the relative expression quantity of goal gene in each group of sample.3 parallel holes established by each sample, establish the blank not adding template simultaneously.CFX96 real-time fluorescence quantitative PCR instrument (Bio-Rad company) increases, and uses SsoFast ^tM supermix detects.

According to gene sequencing result, utilize software Primer Premier 5.0, Oligo6.67 to design Real time PCR and detect primer, product size at about 200bp as well.

(1) UL18Real time PCR detects primer:

UL18Real detects primer upstream: 5'-TTTCCCGTTCCGCTTCCA-3';

UL18Real detects primer downstream: 5'-AGAGGCGACTCCCGTTGTAG-3';

Product length 145bp.

(2) GAPDH Real time PCR detects primer:

GAPDH Real detects primer upstream: 5'-CCCACTCCTCCACCTTTGAC-3';

GAPDH Real detects primer downstream: 5'-TCTTCCTCTTGTGCTCTTGC-3';

Product length 182bp.

(3) reaction system:

(4) reaction conditions:

①95℃for 1min；

②95℃for 5s；

③58℃for 5s+Plate read；

④Go to step②for 39more times；

⑤Melting Curve from 65℃to 95℃，read every 0.2℃，hold 3s+Plate read。

Bio-Rad CFX Manager software analysis result, calculates each sample group goal gene relative expression quantity.

Virogene UL18 detection primer and GAPDH detect primer and carry out pcr amplification to cDNA sample respectively, the product amount of the real-time detection reaction of SYBR Green Ι incorporation methods.Utilize the CT value of Bio-Rad CFX Manager software detection each sample and calculate relative expression quantity.

, not there is non-specific amplification in Real time PCR melting curve (see Fig. 3).Adopt and compare the relative expression levels R (see Fig. 4) that Ct method calculates goal gene.1-R calculates siRNA181, siRNA182, siRNA183 silence efficiency to UL18 gene and is respectively 29.78%, 64.15% and 78.56%.

2, Flow cytometry siRNA silence efficiency

(1) pUL18-EGFP-N1 expressing fusion protein recombinant plasmid preparation: after HSV-1 vero cells infection 48h, Trizol method extracted total RNA, reverse transcription synthesis cDNA first chain.Take cDNA as template, pcr amplification UL18 goal gene.Reaction product 1% agarose gel electrophoresis is identified, reclaims and purified pcr product UL18 fragment.Inoculation contains intestinal bacteria (Escherichia coli) the DH5 α (bacillus coli DH 5 alpha is commercially available bacterial strain) of pEGFP-N1 plasmid, 37 DEG C of overnight incubation.Plasmid is little takes out test kit (purchased from OMEGA company) extracting pEGFP-N1 plasmid; With PstI and HindIII double digestion PCR primer UL18 fragment and expression vector pEGFP-N1,37 DEG C of water-bath 6h, digestion products carries out 1% agarose gel electrophoresis, and glue reclaims digestion products.The enzyme of the UL18 fragment of PCR primer is cut back to close product to cut back to close product with the enzyme of expression vector pEGFP-N1 and be connected 16h in 16 DEG C, product conversion will be connected in bacillus coli DH 5 alpha competent cell, by transformed bacteria liquid coated plate overnight incubation, picking mono-clonal carries out bacterium liquid PCR to be identified, the positive colony that bacterium liquid PCR identifies is carrying out bacterium liquid enlarged culturing, extract recombinant plasmid, carry out double digestion qualification and order-checking qualification.

Qualification result shows the pUL18-EGFP-N1 expressing fusion protein recombinant plasmid of successfully restructuring, carries out extracting in enormous quantities, and after the pUL18-EGFP-N1 recombinant plasmid of extracting surveys plasmid concentration and purity ,-20 DEG C save backup.

(2) by after siRNA and pUL18-EGFP-N1 recombinant plasmid cotransfection 48h, inhale and abandon culture supernatant, cold PBS washes once, and every hole adds 200 μ L trypsin digestion cells; Pancreatin is abandoned in suction, and every hole adds the cold PBS of 600 μ L and blows and beats cell, is transferred to 1.5mL EP and manages, the centrifugal 3min of 1000r/min.Carefully abandon supernatant, the PBS re-suspended cell that 600 μ L are cold, flow cytometer detects every porocyte average fluorescent strength (MFI) and luciferase expression average positive cell rate a, calculate cell total fluorescence intensity (TFI), i.e. TFI=10000 × MFI × a (PLSCONFM).Compared with negative control siRNA N.C (siN.C) porocyte, quantitative analysis every porocyte UL18-EGFP fluorescence relative expression intensities (relative fluorescence intensity, RFI), calculates siRNA silence efficiency.

Flow cytomery result (Fig. 5), calculates cell total fluorescence intensity.With singly turn pUL18-EGFP-N1 group and compare, siRNA N.C negative control group fluorescence intensity and its there was no significant difference (p>0.05), siRNA experimental group fluorescence intensity has with it significant difference (p<0.01) (see Fig. 5).Compared with siRNA N.C negative control, siRNA181, siRNA182, siRNA183 silence efficiency to UL18 gene is respectively 80.1%, 94.76% and 91.83%.

3, the experiment of plaque subtrahend detects the restraining effect that siRNA breeds HSV-1

1. the Vero cell dilution of trysinization is 1.8 × 10 by cell growth medium ⁵cells/mL, 1mL/ implant in hole 24 porocyte culture plates, are placed in 37 DEG C, 5%CO ₂incubator is cultured to cell 70% and converges; 2. getting 20pmolsiRNA is diluted in 50 μ L OPTI-MEM transfection nutrient solutions, mixing; 3. get 1 μ LLipofectamineRNAiMAX and be diluted in 50 μ L OPTI-MEM transfection nutrient solutions, incubated at room 5min; 4. 2. step is mixed with step transfection nutrient solution 3., form transfection cocktail, incubated at room 20min; 5. get 24 good in advance porocyte culture plates, inhale and abandon culture supernatant, every hole adds 400 μ L OPTI-MEM transfection nutrient solutions and 100 μ L transfection cocktail, and vibration plate mixing gently, in 37 DEG C, 5%CO ₂incubator continues to cultivate; 6. after transfection 4h, inhale and abandon transfection cocktail, add HSV-1 viral dilution liquid (30PFU/ hole) 100 μ L, 37 DEG C, 5%CO2 incubator continues to cultivate, rocker is once gently for every 15min; 7. after virus infection 2h, inhale and abandon virus liquid, cold PBS solution washes one time, and the carboxymethyl cellulose adding 0.1% (w/w) covers nutrient solution 1mL, 37 DEG C, 5%CO ₂incubator continues to cultivate; 8., after virus infection 72h, inhale and abandon carboxymethyl cellulose covering liquid, add 10% (v/v) formaldehyde and fix 15min; 9. inhale and abandon formalin, add 500 μ L 1% (w/v) violet staining liquid; After dyeing 20min, tap water is slowly rinsed well; 10. plaque is counted, according to the plaque test inhibiting rate of formulae discovery siRNA.

Plaque result shows: compare with virus control group, siRNA for HSV-1 virus UL18 gene effectively can suppress the formation of virus plaque, plaque number and virus control have significant difference (P<0.01), and negative control siRNA N.C (siN.C) forms not impact (P>0.05) (see Fig. 6) to virus plaque.SiRNA181, siRNA182, siRNA183 form inhibiting rate to HSV-1 virus plaque and are respectively 58.02%, 79.39%, 79.39% (table 3).

The inhibiting rate that table 3siRNA disturbs UL18 gene pairs virus plaque to be formed

V is HSV-1 contrast, * P>0.05, * * P<0.05, * * * P<0.01, ^△represent that siRNA N.C negative control group is more different in nature than indifference with HSV-1 control group.

4, siRNA is on the impact of intracellular virus number of particles after HSV-1 cells infected

By the Vero morphocytology change of HSV-1 virus infection after transmission electron microscope observing siRNA effect, find that virus control group and siRNA N.C (siN.C) negative control group cell surface adsorb more virion, containing a large amount of viral capsid in nucleus, and siRNA effect papova content obviously reduces, few virion (Fig. 7) in core.

The application of embodiment 5siRNA

SiRNA double-strand sequence provided by the present invention and derivative modifier can be used for medicine or the preparation of preparing prevention or treatment HSV-1 and the disease relevant to HSV-1 virus infection.This siRNA double-strand sequence is entered into after in organism by certain mode, the expression of suppression HSV-1 disease-related capsid protein that can be efficient, special, related gene is occurred reticent, thus the expression of target gene is effectively knocked out, finally reach treatment and prevention HSV-1 and the disease object relevant to HSV-1 virus infection.Target of the present invention is clear and definite, successful.

Topmost purposes comprises: make eye drop, can be used for prevention and therapy HSV-1 and infects the eye illness caused; As basting agent, can be used for prevention and therapy HSV-1 and infect the herpes labialis, the genital herpes that cause; As injection, can prevention and therapy HSV-1 latent infection and recurrence etc.

Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; change, the modification done under other any does not deviate from spirit of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.

Claims (9)

1. the siRNA of target HSV-1 virus UL18 gene, is characterized in that: described siRNA is one in siRNA181, siRNA182 or siRNA183 or at least two kinds:

SiRNA181: positive-sense strand: 5'-CUCAACGUGCUGUACUACAAC-3';

Antisense strand: 5'-UGUAGUACAGCACGUUGAGGU-3';

SiRNA182: positive-sense strand: 5'-CCACCAUCAUCCUUACGCUAA-3';

Antisense strand: 5'-AGCGUAAGGAUGAUGGUGGUU-3';

SiRNA183: positive-sense strand: 5'-CCCGUUAUACGCUAUCCCUAA-3';

Antisense strand: 5'-AGGGAUAGCGUAUAACGGGGG-3'.

2. the siRNA of target HSV-1 virus UL18 gene according to claim 1, is characterized in that: described siRNA is carried out end modified or base modification.

3. the siRNA of target HSV-1 according to claim 2 virus UL18 gene, is characterized in that: described end modifiedly to modify for FAM, CY3, vitamin H, amino, cholesterol, phosphate or S-S.

4. the siRNA of target HSV-1 virus UL18 gene according to claim 2, is characterized in that: described base modification is that 2' methoxy is modified, 2' fluoro is modified or thio-modification.

5. the siRNA of the target HSV-1 virus UL18 gene described in any one of Claims 1 to 4 is suppressing the application in UL18 genetic expression.

6. the siRNA of the target HSV-1 virus UL18 gene described in any one of Claims 1 to 4 preparation prevention or treatment HSV-1 and with the application in the medicine of HSV-1 virus infection relative disease or preparation.

7. application according to claim 6, is characterized in that: described medicine or preparation are eye drop.

8. application according to claim 6, is characterized in that: described medicine or preparation are basting agent.

9. application according to claim 6, is characterized in that: described medicine or preparation are injection.