CN109762813A - A kind of synthesis of EGS nucleic acid drug for resisting influenza virus - Google Patents

Abstract

The invention discloses a kind of synthesis of EGS nucleic acid drug for resisting influenza virus, it is the following steps are included: step 1, the determination in the target sequence region that popularity common cold virus gene mRNA is cut under EGS guidance RNase P；Step 2, the design of EGS: the structure of EGS usually combines three parts of region sequence to form by 5 ' in conjunction with region sequence, the T stem ring of tRNA and extra loop region sequence and 3 '；Step 3, EGS's is artificial synthesized: designed EGS sequence synthesized, it is spare；The result of synthesis: step 4 according to sequence general formula 5'-U/CGNNNNNNU-3', searches 100 or so potential point of contacts in eight genetic fragments of influenza virus.The present invention is chemically modified certain groups to improve its stability and import efficiency, greatlys improve the efficiency of EGS genomic medicine, strengthens the broad spectrum activity of anti influenza.

Description

A kind of synthesis of EGS nucleic acid drug for resisting influenza virus

Technical field

The present invention relates to a kind of synthesis of EGS nucleic acid drug for resisting influenza virus.

Background technique

Influenza virus (Influenza virus) is the pathogen of influenza (abbreviation influenza).For a long time with Come, influenza is always to endanger the severe infection disease of human health and public security, and annual economic loss resulting from is also very It is huge.The single-stranded RNA virus that Influenza Virus orthomyxoviridae family influenza A virus belongs to.Typical virion is spherical in shape, has Cyst membrane, diameter are 80 ~ 120 nanometers, there is uneven in length filamentous particle individually, and cyst membrane surface is covered with compact arranged fibre and dashes forward, Respectively viral hemagglutinin and neuraminidase protein.

According to the albuminous difference of NP, influenza virus can be divided into A, tri- type of B, C.According to the antigenic difference of HA and NA, Different subtype can be divided into, hitherto it is found that 16 kinds of HA(H1 ~ H16), 10 kinds of NA(N1 ~ N10), any HA is in conjunction with any NA It is afterwards a kind of blood serum subtype.In nature, influenza virus lasting stream among the animals such as people and pig and horse, especially birds Row.Influenza virus gene group is segmented, sub-thread strand RNA, shares 8 independent RNA segments.Be separately encoded polymerase PB1, PB2, PA, hemagglutinin (HA), nucleocapsid protein (NP), neuraminidase (NA), matrix protein l and M2, non-structural protein NS 1 And NS2.

The polymerase (Polymerase) of influenza virus is made of PB2, PB1 and PA, is compiled by viral RNA segment (1- 3) Code, molecular weight are followed successively by 87KD.96KD.85KD.PB2 is identified in the initial phase of viral mRNA transcription and is incorporated in 6 ' end I On type cap-like structure, in addition it participates in the cutting of host mRNA cap-like structure there are also the activity of restriction enzyme.PB1, in disease It is allowed to gradually extend after malicious mRNA synthesis starting.PA, with chain together with PB1, PB2 in transcription of viral RNA and reproduction process Extend and move, be primarily involved in the duplication of viral RNA, collectively forms RNA polymerase complex with PB1, PB2.In viral RNA Play protein kinase or unwindase in synthesis process.

Hemagglutinin (Hemagglutinin, HA) is to constitute prominent one of the main component of influenza virus cyst membrane fibre, by segment 4 Coding.It is to be attached to virus on cell in conjunction with the cell receptor of host's sialic acid etc, virus is helped to penetrate the cell of host Film simultaneously changes antigenicity, to escape the monitoring of host immune system.

Nucleoprotein (Nucleoprotein, NP) is 60KD by the structural proteins molecular weight that segment 5 encodes, it is viral core clothing The primary protein component of shell.With type specificity.

The fibre that neuraminidase (Neuraminidase, NA) also constitutes virus envelope is prominent, exists in cluster.NA is a kind of saliva Liquid acid enzyme, can identify the sialic acid residues of cell surface influenza viral receptor end, and virus is made to be able to enter cell, and NA's is another One function is to clear up channel for the virion to be sprouted, prevents virion aggregation, be conducive to virion maturation and Release.

There are two types of matrix protein l, M2 for stromatin (Matrixproteins, M).M1 is by 252 amino acid residue groups At it is one kind that content is most in all protein ingredients that molecular weight, which is about 26KD, constitutes the matrix membrane of virus, has type special Property.M2 is made of 97 amino acid residues, and molecular weight is about 15KD.It is a kind of transmembrane protein, is present in the form of the tetramer On the cell membrane of infection cell.

Non-structural protein (Nonstructural Proteins, NS) has kind of non-structural protein i.e. NS1 and NS2, molecular weight Respectively 25KD and 12KD, NSl are synthesized early stage infection, and NS2 is synthesized in the later period of infection.NS1 albumen is binding protein, it Have the function of that the mRNA with poly (A) structure is inhibited to transport from inside to outside from nucleus.NS2 is called consideration convey and goes out albumen (NEP), is Phosphorylated protein directly participates in the process that progeny virus RNP is released from virocyte.

Main problem existing for resisiting influenza virus chemicals includes drug resistance, toxic side effect and alkane amine drug pair at present Type B influenza is without three aspect of effect.

It is domestic at present mainly to develop resisiting influenza virus nucleic acid drug using RNA perturbation technique.Sichuan University Yang Xiaofang, Zhao Pei et al. studies the effect of siRNA recombinant plasmid anti-influenza A virus, and it is multiple to further demonstrate RNA interference infected by influenza The inhibiting effect of system, Jiangxi Medical College Liu is luxuriant and bdautiful et al. to have studied anti-influenza virus medicament and Apoptosis, and discovery anti-current is susceptible Cytotoxic drug is other than Derivatives of Adamantane, and there are also influenza viruses by blocking drugs, melanin etc..Chinese People's Liberation Army's military affairs doctor The Wang Shengqi et al. of subject institute has invented a kind of anti-meaning nucleotide of resisiting influenza virus for targeting apoptosis inducing factor, and discovery should Drug can effectively inhibit cytopathy caused by H1N1, H3N2, H5N1 type influenza virus, and Chinese Center for Disease Control and Prevention is high Treasure et al. has invented a kind of siRNA sequence for directing towards influenza B virus nucleocapsid gene forever, and influenza B can be effectively suppressed Duplication and infection of the virus in cell and animal model.In conclusion domestic seen a variety of resisting using RNA interference as theoretical basis The report of influenza virus drug, but have no using EGS-RNA technology as theoretical basis, have the characteristics that high efficiency, broad spectrum activity it is anti- The report of influenza nucleic acids drug development.

Summary of the invention

The present invention provides a kind of synthesis of EGS nucleic acid drug for resisting influenza virus, it is in analysis influenza virus EGS is designed on the basis of PB1 gene order, iii vitro chemical synthesizes EGS, and is chemically modified certain groups to improve it Stability and importing efficiency, greatly improve the efficiency of EGS genomic medicine, strengthen the broad spectrum activity of anti influenza.

The invention adopts the following technical scheme: a kind of synthesis of EGS nucleic acid drug for resisting influenza virus, it includes Following steps:

Step 1, the target sequence region that popularity common cold virus gene mRNA is cut under EGS guidance RNase P It determines: determining that potential point of contact in target RNA, the high efficiency cutting site of target RNA have following characteristics, i.e.-the 1 of point of contact should be divided with+1 Not Wei pyrimidine (U/C) and guanine (G) ,+8 of point of contact should preferably be uracil (U), therefore, according to 5 '-U/ of sequence general formula CGNNNNNNU-3 ' searches potential point of contact in influenza virus PA, PB1, M1 gene；

Step 2, the design of EGS: the structure of EGS usually by 5 ' combine region sequence, the T stem ring of tRNA and extra loop region sequence and 3 ' combine three parts of region sequence to form, wherein T stem ring and extra loop region sequence are fixed, partial sequences of whole EGS It is selected from people tRNA, 5 ' combined areas and 3 ' combine region sequence then not identical in different EGS, A type stream is found in GeneBank The conserved sequence of Influenza Virus clinical strain's target sequence PA, PB1 and M1 gene, in the fixed situation in potential point of contact, according to this Sequence near point of contact, according to the principle of base pair complementarity, design is directed to the EGS combination region sequence at the point of contact, in design It should be noted that -1 of point of contact cannot be with the base pairing of EGS: should be spaced between sequence target RNA complementary with the two sides EGS combined area 2nt；EGS combination region sequence has 12nt at least to guarantee the specificity of complementary combination as far as possible, designed whole EGS, such as The combined area sequence length of EGS E-PA, E-PB1 and E-M1 are 14-16nt, and 5 ' combined areas and 3 ' combine the length of region sequence It is respectively 7-9 nt；

Step 3, EGS's is artificial synthesized: designed EGS sequence being synthesized, when synthesis adds 2 '-O-methyl chemistry Modification, while the corresponding control EGS (Control EGS, C-EGS) for designed EGS has also been synthesized as control examination It tests, is dissolved EGS lyophilized products with sterile tri-distilled water, and be diluted to 100uM, packing is stored in -20 DEG C, -80 DEG C, spare；

Step 4, the result of synthesis: according to sequence general formula 5'-U/CGNNNNNNU-3', in eight genetic fragments of influenza virus 100 or so potential point of contacts are searched, in addition, there are the conserved motifs of three bases " TTC " in the T ring in EGS, if changed Become conserved motifs, guidance cleavage activity will be substantially reduced, and on the basis of normal EGS, respectively be kept the T environmental protection in each EGS Motif " TTC " all becomes " AAG ", using the control EGS (controlEGS, C-EGS) as corresponding EGS.By In vitro digestion Experiment screening is respectively designated as E-PA, E-PB1, E-M1 to 3 EGS with obvious cleavage activity, they are located at PA, Corresponding EGS, according to the sequence near at these three sites, is designed in the 15 of PB1, M1 gene by 1224,222,

The sequence of EGS in step 4 of the present invention are as follows:

Number	EGS title	EGS sequence
			1	E-PA	5-GGUUAACAUUGAAGGUGCGGUCUCCGCGCGCAGGUUCAAAUCCUGCUUGUCGCACCA-3
2	E-PB1	5’-GUAUUGAAGGUGCGGUCUCCGCGCGCAGGUUCAAAUCCUGCGCCCAUCACCA-3’
			3	E-M1	5’-GCAAAGCGCGUGCGGUCUCCGCGCGCAGGUUCAAAUCCUGCACGCUGCACCA-3’

By the T ring conserved motifs " UUC " becomes " AAG " in three EGS in step 4 of the present invention, as corresponding control EGS (C33333-EGS), the simultaneously synthesizing EGS for herpes simplex virus type (HSV-1) TK gene, E-TK are as control Its sequence table of sequence are as follows:

Number	EGS title	EGS sequence
			1	C-PA	5’-GGUUAACAUUGAAGGUGCGGUCUCCGCGCGCAGGAAGAAAUCCUGCUUGUCGCACCA-3
2	C-PB1	5’-GUAUUGAAGGUGCGGUCUCCGCGCGCAGGAAGAAAUCCUGCGCCCAUCACCA-3’
			3	C-M1	5’-GCAAAGCGCGUGCGGUCUCCGCGCGCAGGAAGAAAUCCUGCACGCUGCACCA-3’
4	E-TK	5-GCUACGUCGGUGCGGUCUCCGCGCGCAGGUUCAAAUCCUGCCGCAGACACCA-3

The invention has the following advantages: after using above technical scheme, RNase P of the proposed adoption of the present invention based on nucleic acid Therapeutic strategy, design are directed to the EGS of influenza virus PB1 gene, are analyzing according to EGS design principle by RNA analysis software EGS is designed on the basis of influenza virus PB1 gene order, iii vitro chemical synthesizes EGS, and is chemically modified to certain groups To improve its stability and import efficiency.Through liposome, the gene interference that can be used for clinical treatment influenza A is developed EGS drug, have the characteristics that it is special, efficiently, without obvious toxic-side effects, while gene inhibits, and greatlys improve EGS genomic medicine Efficiency, strengthen the broad spectrum activity of anti influenza.Resisiting influenza virus EGS nucleic acid drug of the invention has special, efficient, the obvious poison of nothing The characteristics of side effect, in vitro in Antiviral breeding, influenza virus target gene and influenza virus virus titer divide EGS nucleic acid drug It Xia Jiang 99.1% and 99.6%；EGS nucleic acid drug infected by influenza inhibitory activity in Mice Body reaches 99.7%, protective effect Reach 100%, safety 100%.

Detailed description of the invention

Fig. 1 is potential cleavage site schematic diagram in PA, PB1, M1 gene mRNA of the present invention.

Fig. 2 is the Sketch of secondary structure figure of PA, PB1, M1 gene mRNA/EGS compound of the present invention.

Fig. 3 is EGS of the present invention to PA, PB1, M1 gene mRNA In vitro digestion effect picture.

Fig. 4 is that EGS of the present invention inhibits influenza activity contrast schematic diagram into the cell.

Fig. 5 is for EGS small-molecule drug of the present invention to the inhibitory activity contrast schematic diagram of virus in Mice Body.

Fig. 6 is protectiveness effect figure of the EGS of the present invention to mouse.

The staging that Fig. 7 drug of the present invention influences righting reflex.

Fig. 8 is the staging that drug of the present invention influences passive state.

Fig. 9 is that present invention tracer method combination HPLC method measures the blood concentration-time song of EGS in rats Line simultaneously calculates main pharmacokinetic parameter experiment flow figure.

Specific embodiment

The present invention provides a kind of synthesis of EGS nucleic acid drug for resisting influenza virus, it the following steps are included:

Step 1, the target sequence region that popularity common cold virus gene mRNA is cut under EGS guidance RNase P It determines: determining that potential point of contact in target RNA, the high efficiency cutting site of target RNA have following characteristics, i.e.-the 1 of point of contact should be divided with+1 Not Wei pyrimidine (U/C) and guanine (G) ,+8 of point of contact should preferably be uracil (U), therefore, according to 5 '-U/ of sequence general formula CGNNNNNNU-3 ' searches potential point of contact in influenza virus PA, PB1, M1 gene；

Step 2, the design of EGS: the structure of EGS usually by 5 ' combine region sequence, the T stem ring of tRNA and extra loop region sequence and 3 ' combine three parts of region sequence to form, wherein T stem ring and extra loop region sequence are fixed, partial sequences of whole EGS It is selected from people tRNA, 5 ' combined areas and 3 ' combine region sequence then not identical in different EGS, A type stream is found in GeneBank The conserved sequence of Influenza Virus clinical strain's target sequence PA, PB1 and M1 gene, in the fixed situation in potential point of contact, according to this Sequence near point of contact, according to the principle of base pair complementarity, design is directed to the EGS combination region sequence at the point of contact, in design It should be noted that -1 of point of contact cannot be with the base pairing of EGS: should be spaced between sequence target RNA complementary with the two sides EGS combined area 2nt；EGS combination region sequence has 12nt at least to guarantee the specificity of complementary combination as far as possible, designed whole EGS, such as The combined area sequence length of EGS E-PA, E-PB1 and E-M1 are 14-16nt, and 5 ' combined areas and 3 ' combine the length of region sequence It is respectively 7-9 nt；

Step 3, EGS's is artificial synthesized: designed EGS sequence being synthesized, when synthesis adds 2 '-O-methyl chemistry Modification, while the corresponding control EGS (Control EGS, C-EGS) for designed EGS has also been synthesized as control examination It tests, is dissolved EGS lyophilized products with sterile tri-distilled water, and be diluted to 100uM, packing is stored in -20 DEG C, -80 DEG C, spare；

Step 4, the result of synthesis: according to sequence general formula 5'-U/CGNNNNNNU-3', in eight genetic fragments of influenza virus 100 or so potential point of contacts are searched, in addition, there are the conserved motifs of three bases " TTC " in the T ring in EGS, if changed Become conserved motifs, guidance cleavage activity will be substantially reduced, and on the basis of normal EGS, respectively be kept the T environmental protection in each EGS Motif " TTC " all becomes " AAG ", using the control EGS (controlEGS, C-EGS) as corresponding EGS.By In vitro digestion Experiment screening is respectively designated as E-PA, E-PB1, E-M1 to 3 EGS with obvious cleavage activity, they are located at PA, The 15 of PB1, M1 gene, 1224,222, distribution designs phase as shown in Figure 1, according to the sequence near at these three sites The EGS answered.

The sequence of EGS in step 4 of the present invention are as follows:

Number	EGS title	EGS sequence
			1	E-PA	5-GGUUAACAUUGAAGGUGCGGUCUCCGCGCGCAGGUUCAAAUCCUGCUUGUCGCACCA-3
2	E-PB1	5’-GUAUUGAAGGUGCGGUCUCCGCGCGCAGGUUCAAAUCCUGCGCCCAUCACCA-3’
			3	E-M1	5’-GCAAAGCGCGUGCGGUCUCCGCGCGCAGGUUCAAAUCCUGCACGCUGCACCA-3’

T ring conserved motifs " UUC " in three EGS are become into " AAG " in step 4 of the present invention, as corresponding control EGS (C33333-EGS), the simultaneously synthesizing EGS for herpes simplex virus type (HSV-1) TK gene, E-TK are as control sequence Arrange its sequence table of are as follows:

Number	EGS title	EGS sequence
			1	C-PA	5’-GGUUAACAUUGAAGGUGCGGUCUCCGCGCGCAGGAAGAAAUCCUGCUUGUCGCACCA-3
2	C-PB1	5’-GUAUUGAAGGUGCGGUCUCCGCGCGCAGGAAGAAAUCCUGCGCCCAUCACCA-3’
			3	C-M1	5’-GCAAAGCGCGUGCGGUCUCCGCGCGCAGGAAGAAAUCCUGCACGCUGCACCA-3’
4	E-TK	5-GCUACGUCGGUGCGGUCUCCGCGCGCAGGUUCAAAUCCUGCCGCAGACACCA-3

The effect of the invention further illustrated below by following experiment.

One, the RNase P that EGS is mediated carries out extracellular cutting experiment to target gene mRNA.

1, the clone of influenza virus PA, PB1, M1 gene and in-vitro transcription: building DNA profiling expression PA, PB1, M1 mRNA sequence passes through T7 RNA polymerase in-vitro transcription kit, popularity common cold virus PA, PB1, M1mRNA sequence It is transcribed in vitro.Pancreas DNA enzymatic I (1 mg/ml) of the lul without RNA enzyme, mixing is added in end of reaction, every 50u1 transcription volume Uniformly in 37 DEG C of incubation 15-30min.The water that 150 ul are polluted without RNA enzyme is added, with isometric phenol/chloroform/isoamyl alcohol (25: 24:1) extract.20ul 3M NaAC is added, the dehydrated alcohol of 3 times of volumes (660ul) is added, ice 1h after mixing, 4 DEG C 12000rpm is centrifuged 25min.Carefully siphon away supernatant, 75% ethanol washing of 300u1 precipitating is added in every pipe, at 4 DEG C 12000rpm from Heart 15min, carefully siphons away supernatant, and eppendorf pipe is placed in drying (about 15min) in super-clean bench, finally plus sterile The dissolution of DEPC water, -20 DEG C save backup.

2, it the extraction and activity identification of RNase P: is trained under the conditions of 37 DEG C, 5%C02 with DMEM culture medium (containing 10% FBS) Support people's cell (such as: HeLa Cells).When the cell density in culture bottle about 70%, abandons supernatant and washed once with PBS, Then cell is gently scraped with cell scraper, 2500rpm is centrifuged 1Omin and collects cell.It is freezed in cell precipitation postposition liquid nitrogen, it Afterwards in -80 DEG C, natural thaw.Add the PBS of the MgC12.6H2O containing 1g in right amount, 4 DEG C, 2500rpm is centrifuged 10min, abandons supernatant； Cell precipitation adds the buffer solution A (what are the gradients) of 4 times of cell volumes to be resuspended;Cell suspension is in trash ice 30min is stood, final concentration of 0.1% Nonidet (NP-40) is then added；It is homogenized rapidly with homogenizer 20 times, makes cell Cracking.

Cell pyrolysis liquid removes organelle and cell fragment with the centrifugal force 1Omin (4 DEG C) of 100000g.By supernatant Liquid is added slowly to flow into the DEAE-Sepharose Fast-flow column of oneself balance (2.5X56cm) to sample almost all When, close peristaltic pump about 30min.Then, then with the buffer solution A of the KCl containing 100mM~5OOmM (10mM Tris-HC1, PH7.5,2.5mM MgCl2,2mM DTT, 5mM KC1, pefabloc) row gradient elution.It is monitored and is eluted by Ultraviolet Detector Protein peak, collect each peak albumen.Finally, and using concentration centrifuge tube that it is concentrated with 8000rpm centrifugation 30min.RNase P is living Property measurement use tRNA substrate cleavage reaction.

3, extracellular cutting experiment: the isotope [32P] that influenza virus PA, PB1, M1 gene is transcribed in vitro is marked The RNA of note is mixed with equimolar each EGS segment;It is then respectively adding the RNase P of extraction, reacts 30min at 37 DEG C；With 8% urea-denatured PAGE (urea containing 7M) separation, by analyzing each EGS to the cutting situation of target gene mRNA.

4, under the effect of T7 RNA polymerase body, the influenza virus of isotope [32P] label result: is transcribed in vitro PA, PB1, M1 gene mRNA.The mRNA and equimolar each EGS that influenza virus PA, PB1, M1 gene is transcribed in vitro Segment mixing;It is then respectively adding the RNase P of extraction, reacts 30min at 37 DEG C；It (is urinated containing 7M with 8% urea-denatured PAGE Element) separation, by analyzing each EGS to the cutting situation of target gene mRNA.PA, PB1, M1 gene mRNA and corresponding EGS second level Tactic pattern figure, is shown in Fig. 2.

The extracellular cleavage activity of three kinds of EGS guidance RNase P is as shown in Figure 3.Swimming lane 1,3,5 is only to add substrate and RNase P, and the negative control of EGS is not added;Swimming lane 2,4,6 adds EGS and substrate and RNase P simultaneously.It can be seen from the figure that three EGS(EGS-PB1, EGS-PA, EGS-M1) RNase P is able to guide to purpose mRNA implementation effectively cutting and is had very high extracellular Guide cleavage activity.

It compares EGS (C-EGS), for the EGS of cytomegalovirus (HCMV) TK gene, E-TK is not guided in vitro The activity of RNase P cutting.

Two, the RNase P Antiviral breeding in the cell that EGS is mediated.

1, as MDCK long to 80%~90%, the culture medium in hole, D- infection of the influenza virus to MDCK: are absorbed Hanks liquid washs one time；Diluted virus liquid (Multiplicity of Infection is inoculated with into each culture hole cell (MOI)=1 it), in 37 DEG C, is cultivated under the conditions of 5%CO2.

2, the liposome transfection of EGS: after influenza virus is inoculated with mdck cell 4h.2h lipid before transfection starts Body transfects EGS, absorbs original culture solution, changes with serum free medium.Certain density EGS will be diluted to and liposome mixes Mixing, stands 20min at room temperature；EGS/ liposome complex is added to each hole of corresponding culture plate, 1u1EGS/ liposome/ Hole (~ 0.1 nmol)；Insulating box transfection 6h-8h is put into after jog is even；Transfection finishes, and the MEM culture medium of 2%FBS serum is added, Terminate transfection.

Different time after virus infection (12h after infection, 18 h, for 24 hours, 48h, 72h) harvest infected cell, extract total RNA analyzes EGS by Northern blot to PA, the inhibition situation of PB1 and M1 mRNA expression.

Separately after the virus infection different time (12h after infection, 18 h, for 24 hours, 48h, 72h) harvest infected cell, lead to Measurement virus titer and viral growth curves etc. are crossed to analyze and confirm that EGS popularity common cold virus is inhibited.This reality It tests and is repeated 3 times, 3 experimental result average values is taken to be analyzed.

3, result: in order to further confirm the EGS selected by In vitro digestion testing sieve in antiviral effect intracellular.

As MDCK long to 80%~90%, the culture medium in hole is absorbed, D-Hanks liquid washs one time；It is thin to each culture hole It is inoculated with diluted virus liquid (Multiplicity of Infection (MOI)=1) in born of the same parents, in 37 DEG C, is trained under the conditions of 5%CO2 It supports.After influenza virus is inoculated with mdck cell 4h.Respectively by these EGS (E-PA (0.1 nmol), E-PB1 (0.1 ), nmol E-M1 (0.1 nmol), E-PA (0.1 nmol)+E-PB1 (0.1 nmol) and E-PA (0.1 nmol)+E-PB1 (0.1 nmol)+E-M1 (0.1 nmol)) and accordingly compare EGS (C-PA, C-PB1, C-M1, C-PA+C-PB1+C- M1 and E-TK) the good mdck cell of growth conditions is entered by liposome transfection respectively, while being set as transfecting the normal of any EGS Mdck cell does negative control.Different time after virus infection (12h after infection, 18 h, for 24 hours, 48h, 72h) extracting cell Total serum IgE.The cutting situation of each EGS infected by influenza target gene mRNA is analyzed by Northern blot.When measuring each simultaneously Between section virus titer.This experiment is repeated 3 times, and 3 experimental result average values is taken to be analyzed.

As a result, it has been found that (as shown in Figure 4), transfection EGS (E-PA, E-PB1 and E-M1) group is with any EGS's of untransfected Mdck cell control group, transfection control EGS (C-PA C-PB1, C-M1 and E-TK) group are compared, influenza virus target gene MRNA and Influenza virus titer have and obviously reduce.E-PA, E-PB1, E-M1, E-PA+E-PB1 and E-PA+ PAmRNA in the transfection group of E-PB1+E-M1, PB1mRNA, the level and Influenza virus titer of M1mRNA are distinguished after infecting 48h Decline 80.2% (Viral Titer:90.1%), 86.3% (Viral Titer:95.2%), 75.1% (Viral Titer: 85.2%), 95.2% (Viral Titer:99.1%) and 99.1% (Viral Titer:99.6%).And C-PA, C-PB1, C- In the mdck cell control group of any EGS of transfection group and untransfected of M1, C-PA+C-PB1+C-M and E-TK, influenza virus target The level and virus titer of mRNA does not reduce significantly.Show that the inhibiting effect of E-PB1 is all remarkably higher than E-PA and E- M1.E-PA inhibiting effect is taken second place, and E-M1 inhibiting effect is most weak.And E-PA+E-PB1+E-M1, E-PA+E-PB1 synergy pair Much higher than each EGS independent role of inhibiting effect of virus.

Three, the RNase P that EGS is mediated is tested in BALB/c mouse interior resisting virus.

In order to further confirm the EGS antiviral work in animal body by inhibiting Viral experiment to filter out into the cell Property.6-8 week old BALB/c mouse is taken, 110, half male and half female is randomly divided into 11 groups, every group 10.

Every group of mouse is inoculated with influenza A strain (A/Human/Taizhou/09 plants), 5x105 by the way of collunarium (500,000) plaque forming unit (pfu)/mouse.It is later small to each group in a manner of spraying nose respectively to meet malicious 12h Mouse is inoculated with EGS (E-PA, E-PB1, E-M1, E-PA+E-PB1 and E-PA+E-PB1+E-M1) and compares EGS accordingly (C-PA, C-PB1, C-M1, C-PA+C-PB1+C-M1 and E-TK), while spraying nose and doing negative control without the PBS of any EGS. After infecting 24,48 and 72h respectively, the lungs of each group mouse are acquired, the Influenza virus titer of each period is measured.This reality It tests and is repeated 3 times, 3 experimental result average values is taken to be analyzed.

It can be seen from the experiment that the EGS of 4 kinds of dosage of 3 kinds of EGS is safety to BALB/c mouse, after mouse injects EGS After, the items physical signs such as weight, body temperature and control group are without marked difference.

Four, the RNase P that EGS is mediated is in BALB/c to the protective effect of inoculation influenza virus.

1, the inhibitory activity of EGS nucleic acid small molecule infected by influenza in BALB/c mouse: in order to further confirm process The antiviral activity of the intracellular EGS for inhibiting Viral experiment to filter out in animal body.Take 6-8 week old BALB/c mouse, 110 Only, half male and half female is randomly divided into 11 groups, every group 10.

Every group of mouse is inoculated with influenza A strain (A/Human/Taizhou/09 plants) by the way of collunarium, and 500,000 Pfu/ mouse.It meets malicious 12h and gives each group mouse inoculation EGS (E-PA (2 micromol), E- in a manner of spraying nose respectively later PB1 (2 micromol), E-M1 (2 micromol), E-PA (2 micromol)+E-PB1 (2 micromol) and E-PA (2 micromol)+E-PB1 (2 micromol)+E-M1 (2 micromol)) and accordingly compare EGS (C-PA, C- PB1, C-M1, C-PA+C-PB1+C-M1 and E-TK), while spraying nose and doing negative control without the PBS of any EGS.It infects respectively After 24,48,96 and 144h, the lungs of each group mouse are acquired, the Influenza virus titer of each period is measured.This experiment It is repeated 3 times, 3 experimental result average values is taken to be analyzed.

As a result, it has been found that as shown in figure 5, inoculation EGS (E-PA, E-PB1, E-M1, E-PA+E-PB1 and E-PA+E-PB1+ E-M1) group and any PBS control group without any EGS of inoculation, transfection control EGS (C-PA, C-PB1, C-M1, C-PA+ C-PB1+C-M1 is compared with E-TK) group, and Influenza virus titer has and obviously reduces.E-PA , E-PB1 , E-M1 , E- Influenza virus titer declines 93.1%, 95.3% after infecting 96h respectively in the inoculation group of PA+E-PB1 and E-PA+E-PB1+E-M1 , 91.2%, 98.5% and 99.7%.And it the inoculation group group of C-PA, C-PB1, C-M1, C-PA+C-PB1+C-M and E-TK and connects In any PBS control group without any EGS of kind, Influenza virus titer is not reduced significantly.The result shows that E-PB1 Inhibiting effect is all remarkably higher than E-PA and E-M1.E-PA inhibiting effect is taken second place, and E- M1 inhibiting effect is most weak.And E-PA+E-PB1 Inhibiting effect than each EGS independent role higher more of+E-M1, the E-PA+E-PB1 synergy to virus.

2, protective effect of the EGS nucleic acid small molecule to BALB/c mouse inoculation influenza virus: in order to further confirm process Antiviral protection of the EGS for inhibiting Viral experiment to filter out in Mice Body to animal.6-8 week old BALB/c mouse is taken, it is 80, female It is male fifty-fifty, it is randomly divided into 8 groups, every group 10.

Every group of mouse is inoculated with influenza A strain (A/Human/Taizhou/09 plants) by the way of collunarium, and 500,000 Pfu/ mouse.It meets malicious 12h and gives each group mouse inoculation EGS (E-PA (2 micromol), E- in a manner of spraying nose respectively later PB1 (2 micromol), E-M1 (2 micromol), E-PA (2 micromol)+E-PB1 (2 micromol) and E-PA (2 micromol)+E-PB1 (2 micromol)+E-M1 (2 micromol)) and accordingly compare EGS (C-PA+C-PB1 + C-M1 and E-TK), while spraying nose and doing negative control without the PBS of any EGS.Within virus infected mice the latter moon, note Record the survival condition of influenza virus Mice Inoculated.

As a result, it has been found that as shown in fig. 6, using spray nose mode be inoculated with control EGS (C-PA+C-PB1+C-M1 and E-TK) and PBS negative control group mouse without any EGS is all dead within 6 days after virus inoculation;Inoculation E-M1 group mouse exists It is all dead within 11 days after virus inoculation;E-PA group mouse is inoculated with after virus inoculation, it is all dead within 13 days;It connects Kind E-PB1 group mouse is all dead within 16 days after virus inoculation;E-PA+E-PB1 group mouse is inoculated with after virus inoculation, Each dead 1 mouse at 19,20 days respectively finally survives 8;And E-PA+E-PB1+E-M1 group mouse is inoculated in virus inoculation Afterwards, it all survives in 30 days 8.Appoint the result shows that spray rhinovaccination compares EGS (C-PA+C-PB1+C-M1 and E-TK) and is free of The PBS of what EGS does not have any protective effect to mouse, only has part when being individually inoculated with EGS (E-PA, E-PB1 and E-M1) Protective effect, and the protective effect of E-PB1 is apparently higher than both other.E-PA+E-PB1 synergy can then play good guarantor Effect is protected, and E-PA+E-PB1+E-M1 triple combination effect can then play 100% protecting effect.This experiment is repeated 3 times, as a result Unanimously.

Five, pilot process is studied.

1 synthesising process research: using solid phase phosphoramidite triester method, synthesizes RNase P- boot sequence.This method has Efficiently, quickly coupling and the more stable feature of initial reactant.

Phosphoramidite triester method be DNA is fixed on solid phase carrier complete DNA chain synthesis, the direction of synthesis be by What the end 3' of primer to be synthesized was synthesized to the end 5', adjacent nucleotide is keyed by 3' → 5' di-phosphate ester.It will connect in advance The protected nucleotide of active group on solid phase carrier CPG is reacted with trichloroacetic acid；The raw material of synthetic DNA, phosphoramidite Nucleotide monomer is protected, is mixed with activator tetrazole, obtains nucleosides phosphorous acid activated intermediate；Band cap reacts, this short-movie Section can be separated in purifying；Under the action of oxidant iodine, phosphorous acyl form is changed into more stable phosphotriester.It repeats Above step, until the base of required synthesis is connected.By the above synthetic reaction, a complete RNase is formed P- boot sequence molecule.By ammonium hydroxide high-temperature process, the primer being connected on CPG, which is cut, to be come, and completes the synthesis of EGS.

2, purifying process is studied: synthesis in solid state-takes supernatant-HPLC purifying, and the final purity of stoste reaches 99% or more, returns Yield reaches 90%, and by continuous three batches of purification experiments, obtains three batches of stable data.All equal nothings of buffer system The pollution such as DNase, endotoxin.The DNA product of collection is concentrated in freeze drier.Concentration of DNA product G10 Gel filtration chromatography column carries out desalting processing, chromatographic condition are as follows: desalination mobile phase is endotoxin-free, without DNase water, desalination RNA freeze-drying will be collected afterwards, carry out quality testing.

Carry out analysis assessment for research conditions, optimize in the following areas: loading elution requirement needs to refine analysis With optimization；If purpose nucleotide chain cannot meet formulation requirements, a variety of concentration means are used.

3, EGS quality testing is studied

The detection of (1) 15% polyacrylamide gel electrophoresis is observed under gel imaging system after EB dyeing and is taken pictures;Purifying is produced Product carry out the yield that UV detection calculates product.

(2) analytic type ion-exchange high-voltage liquid phase chromatogram analyzes sample purity.Finished product is concentrated with C18, desalination, precipitating.It is heavy RNase P- boot sequence aqueous suspension behind shallow lake, measurement OD260 is quantitative, according to requiring to dispense.

(3) DNA albumen and endotoxin residue detection press 2010 editions annex XII E baterial endotoxin tests of Chinese Pharmacopoeia Method gel method detects DNA purification of samples.

4, preparation: stoste after the assay was approved, takes the RNase P- boot sequence of calculation amount, be added injection endotoxin-free, Water without DNase through filtering, packing, is lyophilized, rolls and cover to obtain the final product, -20 DEG C of finished product preservations.

Six, the acute toxicity test of mouse mainline " EGS nucleic acid antiviral drugs ".

1. research topic purpose: large dosage of " EGS nucleic acid antiviral drugs " is given in a single dose in observation in mouse 24 hours after Toxic reaction and its severity disclose the possible target organ of toxic effect, provide guidance for long term toxication and clinical application.

2. regulation, guideline, document.

2.1 regulations: drug non-clinical research quality management practices (office enable No. 2,2003.9).

2.2 guidelines:

2.2.1 chemicals studies on acute toxicity technological guidance principle, CFDA file, [H] GPT-1, in March, 2005.

2.2.2 treatment biological products non-clinical safety evaluation Technical Review rule, CFDA drug evaluation center.

2.3 bibliography:

2.3.1Guide for the Care and Use of Laboratory Animals, Institute of Laboratory Animal Resources, National Research Council, 1996.

3. test sample and reference substance

3.1 test sample information

EGS nucleic acid antiviral drugs (e.g. E-PB1, C1-PB1).

3.2 reference substance information

Negative control (solvent): 0.9%NaCl injection, achromatism and clarity solution, specification are 250ml/ bottles.

Positive control: bovine serum albumin(BSA) (Albumin, Bovine): pale yellow crystals, 10g/ bottles, 2-8 DEG C of preservation.Face Used time is configured to required concentration with 0.9%NaCl injection liquid.

3.3 test samples are prepared and preparation, mark, preservation and the use of reference substance

3.3.1 test sample is prepared

Preparation method: before administration, by test medicine preparation program and required capacity, appropriate test medicine is weighed, solvent dissolves simultaneously It is diluted to required concentration.

3.3.2 it prepares test sample mark: labelling and be identified outside the test sample container of preparation.

3.3.3 test sample preservation condition and stability after preparing: ready-to-use, prepared test sample is put before use It sets in the foam box equipped with ice bag, gives experimental animal after restoring room temperature when use.

3.4 test samples keep sample: different size respectively leaves and takes 1 when test sample receives.

The keeping and use of 3.5 test samples: being stored in the refrigerator of 2~8 DEG C of test sample room, and the same day is needed to use by the administration same day Test sample amount disposably lead out, the test sample led out be not used before be stored in 2~8 DEG C of refrigerators.

The processing of 3.6 remaining test samples: the test sample that consigner does not break a seal is returned, the remaining test sample to have broken a seal is by useless Gurry processing.

4. experimental animal related data

4.1 experimental system and selection reason:

4.1.1 experimental system: mouse.

4.1.2 select reason: guideline suggestion carries out acute toxicity test using two kinds of animal species.

4.2 experimental animal relevant informations

Kind and strain: ICR mouse.

Grade: SPF grades.

Supplying unit: Shanghai Slac Experimental Animal Co., Ltd..

Quantity and gender: 60, half male and half female.

The range of age when administration starts: 6-7 weeks.

Weight range when administration starts: female: 18 ~ 22g, male: 18 ~ 22g

Remaining animal processing: this test is without remaining animal, without processing.

The environmental condition of 4.3 laboratory animal breeding room

Raising room temperature tolerance band: 20~25 DEG C.

Receptacle temperature difference per day tolerance band :≤3 DEG C.

Receptacle humidity tolerance band: 40-70%.

Rate of ventilation: 10~20 times/h.

Lighting hours: 10 h are bright/and 14h is dark.

4.4 experimental animal cages, cage tool information.

Cage tool type: stainless steel cage tool, specification: 28 cm × 13 cm × 19cm.

Stocking density: single cage raising.

4.5 quarantines and adaptive feeding

Animal is quarantined 1 day after receiving in this center barrier system quarantine room, and the qualified animal of quarantine is then moved into animal feeding Room adapts at least 1 day.

4.6 experimental animal feeds

4.6.1 experimental animal feed essential information.

Feed type: irradiation mouse material.

Source: the Chinese Academy of Sciences, Experimental Animal Center Shanghai, Shanghai Si Laike experimental animal Limited Liability center.

Preservation condition: ventilation, drying, shady place are deposited in.

Sterilizing methods: irradiation sterilization.

Feeding method: free feeding.

4.6.2 feed tests and analyzes: when every batch of feed is put in storage, all leaving and taking the feed nutrition composition point of supplying unit offer Analysis report；Supply of forage unit once a year carries out feed by qualified unit reference national standard (GB 14924.8-2001) It tests and analyzes.

4.7 laboratory animal drinking water

Type: the ultrapure water of ultrapure water machine preparation.

Water feeding method: it freely drinks.

Water quality detection: the detection of animal drinking water every 6 months primary, and detection is according to reference to GB5749-2006, " life is drunk Water hygiene standard ".

4.8 experimental animal paddings

4.8.1 type: corncob.

4.8.2 it sterilization method: is used after high pressure steam sterilization.

4.8.3 padding replacement frequency: twice a week.

5. key instrument equipment

Full automatic urine analyzer: model Aution MAX AX-4280, love section, manufacturer, which carrys out medical electronics (Shanghai), to be had Limit company.

Automatic clinical chemistry analyzer: model Architect c8000, Abbott Laboratories, the U.S., manufacturer

Fully automatic blood cytoanalyze: model XT-2000iv, manufacturer SYSMEX.

Full-automatic blood coagulation analyzer: model Stago STA-R Evolution, manufacturer DIAGNOSTICA STAGO

Electrocardiograph: model ECG-6511, Shanghai photoelectricity medical electronic apparatus company.

6. experimental design

The grouping of 6.1 experimental animals and identification

6.1.1 it experimental animal group technology: is selected according to test method.

6.1.2 it the identification and labeling method of experimental animal: is carried out using earhole number+saturation picric acid (yellow) dyeing Label, group where indicating animal with earhole number, with every group of animal number of picric acid dye marker.

The reasons why administration route of 6.2 test samples and reference substance, volume, time, position, frequency and medication time limit and selection

6.2.1 relevant information is administered

Administration route: using intravenous injection.

Volume: 0.1ml/10g (30ml/kg) is administered.

Medicine-feeding part: tail vein.

Administration time: it is given in 1 minute.

Administration frequency: it is administered once.

Medication: it is administered between each group using the method for isometric(al) not isoconcentration.

6.2.2 administration route, dosage, frequency and the selection reason in medication time limit: the intravenous injection that consigner recommends is clinical Intend with administration route being venoclysis, safe pharmacological testing administration route should be consistent with clinical administration approach.

6.2.3 dose design: see table under 6.2.5.

6.2.4 the reasons why dose design:

A) test medicine maximum concentration is provided according to consigner；

B) capacity 0.3ml/10g weight is administered according to mouse mainline maximum.

6.2.5 group is constituted, dosage and number of animals are shown in Table one

Table one

7. the detection frequency and method of various indexs

7.1 general symptom observations

7.1.1 it observes frequency: agents area being visually observed before the administration same day is administered every time and after administration, other symptoms And beginning eye observation observation 2 times daily in the 2nd day, each 1 time of the morning, afternoon after administration.

7.1.2 number of cases is observed: all experimental animals.

7.1.3 observed content: observe animal death condition and general clinical signs.

7.2 dying or dead animal disposition: if any dead or dying, its dead or preagonal sign should be recorded in detail Shape；The blood sample for acquiring moribund animals as far as possible, is dissected in time.

7.3 weighing body weight

7.3.1 it measures frequency: being surveyed during adaptation 2 times (data as before being administered using the 2nd data), be administered the 2nd, 7,14 day respectively Weighing is primary.

7.3.2 number of cases is measured: all experimental animals.

7.3.3 measuring method: TCS-30D-600 electronic platform scale weighs.

7.4 surplus appetite measurements

7.4.1 measure frequency: measurement is primary weekly.

7.4.2 number of cases is measured: all experimental animals.

7.4.3 measuring method: YP electronic balance weighing.

8. the statistical analysis of data

For all data without statistical analysis, data carry out list, analysis.

The results show that not generating and appointing to mouse after healthy cleaning grade mouse single intravenous injection gives EGS antiviral drugs What abnormal response, does not also cause animal dead, weight also increases with the extension of test period.Inject EGS antiviral drugs Group, injection 0.9%NaCl injection group and the vital sign and weight for not being injected intravenously group mouse are without significant difference (P > 0.05).

Seven, influence of the intravenous injection " EGS antiviral drugs " to central nervous system of mice function

1. research purpose

Observe mouse mainline " EGS antiviral drugs " after to mouse general behavior, spontaneous activity, sports coordination ability shadow It rings, and observes the yellow Jackets of drug and sub-threshold dose whether there is or not synergistic effects.

2. regulation, guideline, document

2.1 regulations: drug non-clinical research quality management practices (office enable No. 2,2003.9)

2.2 guidelines:

2.2.1 chemicals general pharmacology investigative technique guideline, CFDA file, [H] GPT3-1, in March, 2005.

2.2.2 treatment biological products non-clinical safety evaluation Technical Review rule, CFDA drug evaluation center, 2007 January in year.

2.2.3ICH S7A.Safety pharmacology studies for human pharmaceuticals.2001。

2.3 document

2.3.1 Guide for the Care and Use of Laboratory Animals, Institute of Laboratory Animal Resources, National Research Council, 1996.

2.3.2 uncle Yuan person of outstanding talent Liao Ming sun Li Bo, drug toxicology method and technology, Chemical Industry Press, 2007,190-198.

3. test sample and reference substance

3.1 test sample information

EGS nucleic acid antiviral drugs

3.2 reference substance information

Negative control (solvent): 0.9%NaCl injection, achromatism and clarity solution, specification are 250ml/ bottles.

Positive control: bovine serum albumin(BSA) (Albumin, Bovine): pale yellow crystals, 10g/ bottles, 2-8 DEG C of preservation.Face Used time is configured to required concentration with 0.9%NaCl injection liquid.

Preparation method: before administration, by test medicine preparation program and required capacity, appropriate test medicine is weighed, solvent is dilute It releases to required concentration.

3.3.2 prepare test sample mark: the outer label of container for storing test sample is indicated.

3.3.3 test sample preservation condition and stability after preparing:

3.3.4 the quality analysis of test sample: referring to the test sample Quality Analysis Report provided by consigner.

3.4 test samples keep sample: every control gauge lattice leave and take 1.

The keeping and use of 3.5 test samples: test sample is stored in the refrigerator of 4 DEG C of test sample room after receiving, by supplying when use Test product administrative staff provide.

The processing of 3.6 remaining test samples: breaking a seal after requiring experiment according to consigner and the remaining test sample that do not break a seal moves back Return consigner.

4. experimental animal related data

4.1 experimental systems and selection reason:

4.1.1 experimental system: ICR mouse

4.1.2 selecting reason:

1. chemicals general pharmacology experimental technique guideline；

As general pharmacology, this germline animal back scape abundant information；

3. animal is in liberal supply.

4.2 experimental animal relevant informations

4.2.1 kind and strain: ICR mouse.

4.2.2 grade: SPF grades.

4.2.3 supplying unit: Shanghai Slac Experimental Animal Co., Ltd..

4.2.4 quantity and gender: 230, half male and half female.

4.2.5 the range of age when administration starts: 6-7 weeks.

4.2.6 weight range when administration starts: female: 18 ~ 22g, male: 18 ~ 22g.

4.2.7 remaining experimental animal processing: formal test uses 200 mouse, half male and half female, extra mouse and test After mouse cervical dislocation put to death.

4.3 the environmental condition of laboratory animal breeding room

4.3.1 raise room temperature tolerance band: 20~25 DEG C.

4.3.2 receptacle temperature difference per day tolerance band :≤3 DEG C.

4.3.3 receptacle humidity tolerance band: 40~70%.

4.3.4 rate of ventilation: 10~20 times/h.

4.3.5 lighting hours: 10 h are bright/and 14h is dark.

4.4 experimental animal cages, cage tool information

4.4.1 cage tool type: plastics cage tool, specification: 28 cm × 13 cm × 19cm.

4.4.2 stocking density: 5 cages.

4.5 quarantines and adaptive feeding

Animal is quarantined 1 day after receiving in this center barrier system quarantine room, and the qualified animal of quarantine is then moved into animal feeding Room adapts at least 1 day.

4.6 experimental animal feeds

4.6.1 experimental animal feed essential information

Feed type: irradiation mouse material.

Source: the Chinese Academy of Sciences, Experimental Animal Center Shanghai, Shanghai Si Laike experimental animal Limited Liability center.

Preservation condition: ventilation, drying, shady place are deposited in.

Sterilizing methods: irradiation sterilization.

Feeding method: free feeding.

4.6.2 feed tests and analyzes: when every batch of feed is put in storage, all leaving and taking the feed nutrition composition point of supplying unit offer Analysis report；Supply of forage unit once a year carries out feed by qualified unit reference national standard (GB 14924.8-2001) It tests and analyzes.

4.7 laboratory animal drinking water

Type: the ultrapure water of ultrapure water machine preparation.

Water feeding method: it freely drinks.

Water quality detection: the detection of animal drinking water every 6 months primary, and detection is according to reference to GB5749-2006, " life is drunk Water hygiene standard ".

4.8 experimental animal paddings

Type: corncob.

Sterilization method: it is used after high pressure steam sterilization.

Padding replacement frequency: twice a week.

4.9 Laboratory Animal Welfare

Have special IACUC(experimental animal Ethics Committee) be responsible for animal welfare in terms of supervision, examine and instruct work, institute There is the use of animal to ratify by IACUC.This central animals welfare follows " Guide for the Care and Use of Laboratory Animals》[2.3.1]。

5. instrument and equipment

Tired instrument of transfer rod formula: model YLS-4C, manufacturer's Jinan Yi Yan development in science and technology Co., Ltd

Multi-functional autonomic activities recorder: YLS-1A, manufacturer's Jinan Yi Yan development in science and technology Co., Ltd

6. experimental design

The grouping of 6.1 experimental animals and identification

6.1.1 200 animals are divided into 4 parts, every part 50, equal half male and half female.It is respectively completed following experiment content: General behavior, motor coordination, spontaneous activity, subliminal hypnosis.Each experiment sets five groups altogether: solvent control group, low dose group, Middle dose group, high dose group, positive control diazepam group.It is grouped by the weight equilibrium method of dividision into groups, every group of 10 animals, Half male and half female.

6.1.2 it the identification and labeling method of experimental animal: is marked using earhole number+saturation picric acid (yellow) dyeing Note, group where indicating animal with earhole number, with every group of animal number of picric acid dye marker.

6.1.3 the labeling method of experimental animal cage: each equal hanging cage card of cage tool, cage card information includes project number, dynamic Object number, dosage, project responsible person.

The reason of the administration route of 6.2 test samples and reference substance, volume, time, position, frequency and medication time limit and selection By

6.2.1 relevant information is administered

Administration route: using intravenous injection.

Volume: 0.1ml/10g (10ml/kg) is administered.

Medicine-feeding part: tail vein.

Administration time: it is given in 1 minute.

Administration frequency: it is administered once.

Medication: it is administered between each group using the method for isometric(al) not isoconcentration.

6.2.2 the selection reason of administration route, frequency and medication time limit: the test medicine clinic that consigner recommends is quasi- to be used Administration route is venoclysis, and safe pharmacological testing administration route should be consistent with clinical administration approach.

6.2.3 dose design: see table under 6.2.5.

6.2.4 the reasons why dose design:

A) according to this center acute toxicity test in mice.

B) Pharmacodynamics should be equivalent to according to " chemicals general pharmacology investigative technique guideline " low dosage Effective dose, high dose react the requirement being limited not generate serious toxicity.

6.2.5 group is constituted, dosage and number of animals are shown in Table two

Table two

7. the detection frequency and method of various indexs

7.1 general symptom observations

7.1.1 observe frequency: the laundering period is observed twice daily

7.1.2 number of cases is observed: all experimental animals.

7.1.3 observation method: there is symptom that should be described in detail.

7.2 weighing body weight

7.2.1 frequency is measured: before grouping, the administration same day.

7.2.2 number of cases is measured: all experimental animals.

7.2.3 measuring method: electronic balance weighing.

7.3 general behaviors: before observing time is administration, 0.5 after administration, 1,2,3,4 hours.Using Irwin behavior test Method, observation drug is on animal righting reflex, passive state, amyostasia, salivation, nystagmic influence, amyostasia, salivation and eyeball shake By table 3, the influence standards of grading to righting reflex are shown in Fig. 7 for the scoring quivered.Calculate every group of average.

Mouse amyostasia, salivation and nystagmus grade form

0-8 grades can be divided into according to drug effect degree.Method: lifting tail and turn-take 2-3 times, and the different of mouse landing is observed after dishing out Normal posture (side position or under), such five times, is shown in Fig. 8, makes series by figure, 0 is normal condition, and 2,4,6,8 grades are medication Animal is put into a non-natural position afterwards, according to the standard of diagram make it belonging to series.1,3,5,7 grades are between adjacent The position of two interpolars.

7.4 sports coordination ability

Before observing time is administration, 0.5 after administration, 1,2,3,4 hours.Using YLS-4C type tired instrument of transfer rod formula to drug to small Mouse sports coordination ability and muscle tone carry out quantitative analysis.Transfer rod speed is set as 40 revs/min, and each time point records animal The time fallen from transfer rod is converted to score value according in the stick time greater than 3min in terms of 3min: being less than 1min in the stick time Meter 1 divide；2 points are counted (not the including 2min) of 1~2min in the stick time；The stick time 2~3min (not including 3min) 3 points of meter；Divide in meter 4 of the stick time more than or equal to 3min.

7.5 spontaneous activity

Before observing time is administration, 0.5 after administration, 1,2,3,4 hours.Laboratory requires darker light, and test process keeps peace It is quiet；It needs to urinate dung after test every time to remove completely.Each time point records autonomic activities number of the animal in 3min.

7.6 subliminal hypnosis

It determines the subthreshold hypnotic dosage prerun of yellow Jackets: taking test with criticizing mouse 5, with 25 mg/kg intraperitoneal injection 0.125% Nembutal sodium solution, 0.4 ml/20g(dosage is 25mg/kg), animal righting reflex in 15 minutes after observation administration Disappearance 1 minute or more number of animals, if righting reflex loss is more than or equal to 1, then again with 20 mg/kg dosage (reducing by 5 mg/kg) Such as righting reflex loss does not occur for test, then is tested again with 30 mg/kg dosage (increasing by 5 mg/kg), and so on, it finds just The dosage that good righting reflex loss number of animals is 0.

5 minutes intraperitoneal injection sub-threshold dose yellow Jackets after each group administration, observe, record each dosage group and giving penta bar Righting reflex disappears in 1 minute or more mouse number in than 30 minutes after appropriate sodium.Stable group of 15 minutes abdominal cavities note upon administration Sub-threshold dose yellow Jackets are penetrated, observes, record each dosage group righting reflex loss in 30 minutes after giving yellow Jackets In 1 minute or more mouse number.If any with dose-dependent righting reflex loss, can be according to each dosage group righting reflex loss Number of animals calculates ED50 (95% fiducial limit) with Bliss method.Otherwise result is only reported.

7.7 put to death method

Equal cervical dislocation is put to death after all zooperies.

8. the statistical analysis of data

It is for statistical analysis to data with SPSS software.General behavior and movement coordination test data carry out non-parametric test (Kruskal-Wallis H inspection) does Mann-Whitney U inspection between group；Spontaneous activity test data uses single factor test Variance analysis, comparison among groups are tested using Bonferroni method.Subliminal hypnosis: disappear if any with dose-dependent righting reflex It loses, can calculate ED50 (95% fiducial limit) according to each dosage group righting reflex loss number of animals with Bliss method, otherwise only report Accuse result.

Mouse general behavior, autonomic activities and subthreshold hypnotic dosage yellow Jackets Synergism Testing: mouse injection injection EGS Antiviral drugs and excipient, examination has no significant effect mouse autonomic activities, the results show that 0.025,0.075 He 0.25 dosage group of mg/kg tri- administration after 5,15,30 and 60 minutes autonomic activities number with corresponding time negative control group There was no significant difference (P > 0.05) compared to having for 0.9% sodium chloride injection；Three dosage groups and excipient group are to mouse subliminal hypnosis Dose of sodium pentobarbitone is also without synergistic effect, sleep latency and sleeping time and 0.9% chloride injection of negative control group Liquid phase ratio has that there was no significant difference (P > 0.05)；The general behavior of mouse is had no significant effect, with 0.9% chlorine of negative control group Changing sodium injection, there was no significant difference (P > 0.05) compared to having.

Eight, EGS nucleic acid antiviral drugs long term toxicity test

1. mouse repeated doses are administered (14 days) and test

5-6 weeks Wistar mouse is used for tri- groups of tests of A, B, C:

A group: male and female each three, continuous intravenous injection EGS nucleic acid antiviral drugs, the dosage of every animal be 0.03 or 0.15mg/kg/day, two groups of blank controls, wherein one group of injecting normal saline 5ml/kg b. w., another group is not injected.

B group: male and female each three, continuous intravenous injection EGS nucleic acid antiviral drugs, the dosage of every animal is 0.03 Or 0.15 mg/kg/day.Medium ammonia second triol buffer concentration in 0.03mg/kg group is 28.3mg ammonia second triol/ml Distilled water, the medium ammonia second triol buffer concentration in 0.15 mg/kg group are 141.3mg ammonia second triol/ml distilled water.Separately Outside, there are two in blank control, one group of injecting normal saline 5ml/kg b. w., another group of injection 5ml/kg b. w. ammonia second Triol buffer (141.3mg ammonia second triol/ml distilled water).

C group: male and female each 10, continuous intravenous injection EGS nucleic acid antiviral drugs, the dosage of every animal is 0.03 Or 0.09mg/kg/day, 0.03mg/kg/day group inject EGS nucleic acid antiviral drugs and 12.6mgNa2CO3/ml distilled water, 0.15mg/kg/day group injects EGS nucleic acid antiviral drugs and 33.6mgNa2CO3/ml distilled water.There are two blank control group, One group of injection 5ml/kg b. w. physiological saline, the 33.6mgNa2CO3 distilled water of one group of injection 5ml/kg b. w..

Weight twice and food ration are surveyed weekly, observes the general health and behavioral activity of animal daily, are tied in test Hematology, clinical indices, ophthalmoscopy (method), urine qualitative analysis inspection are carried out when beam.All animals will carry out naked eyes and show Micro-inspection.

2. chronic toxicity test

2.1 mouse repeated doses are administered (90 days) and test

A is administration group, and B, C are blank control group

A group: being divided into 3 groups, every group mouse 30 (half male and half female), continuous intraperitoneal administration, dosage be respectively 0.01,0.04 or 0.16mg/kg/day.Each group injectable liquefied composition:

0.01mg/kg/day group --- EGS nucleic acid antiviral drugs+3.3mg Na2CO3/ml distilled water, 0.04mg/kg/ Day group --- EGS nucleic acid antiviral drugs+13.1mg Na2CO3/ml distilled water, 0.16mg/kg/day group --- 306.4mgEGS nucleic acid antiviral drugs+52.5mg Na2CO3/ml distilled water.

B group: mouse 30 (half male and half female), injecting normal saline.

C group: mouse 30 (half male and half female), injection Vehicle, it includes 52.5mgNa2CO3 and distilled water.

After successive administration 90 days, every group of 20 animal (half male and half female) is put to death, remaining animal is after being discontinued 4 weeks Extremely.When on-test, mouse is born 6 weeks, male mouse average weight 123g, female mice average weight 115g.It every weekly check weight and takes the photograph Appetite, daily observe animal general health and behavioral activity, carry out hematology, clinical indices, ophthalmoscopy (method), Urinate qualitative analysis inspection.All animals will carry out naked eyes and micrography.In postmortem check heart, thymus gland, lung, liver,kidney,spleen, The weight of adrenal gland, testis, ovary, seminal vesicle, thyroid gland, hypophysis, brain tissue.To blank control group and two high dose group female mices Kidney carry out not stained slice and micrography.

2.2 test result

It is for statistical analysis to data.

(a) weight and food ration of weight and food ration .A group animal and control group B, the weight and food ration of C group animal Compared to having, there was no significant difference (P > 0.05).

(b) the general health of animal observes the general health and behavioral activity of animal, A group animal daily General health and control group B, the general health of the animal of C group animal compare and have that there was no significant difference (P > 0.05).

(c) hematology of animal and clinical indices carry out hematology, clinical indices, ophthalmoscopy (method), uridine Analysis checks.All animals will carry out naked eyes and micrography.The hematology and clinical indices and control group B of A group animal, C group The hematology of animal has that there was no significant difference (P > 0.05) compared with clinical indices.

(d) heart, thymus gland, lung, liver,kidney,spleen, adrenal gland, testis, ovary, essence are checked in the postmortem postmortem of animal The weight of capsule, thyroid gland, hypophysis, brain tissue.Not stained slice is carried out to the kidney of blank control group and two high dose group female mices And micrography.The postmortem clinical indices and control group B of A group animal, the postmortem clinical indices of C group animal are compared to whether there is or not conspicuousnesses The heart of difference (P > 0.05) A group animal, lung, liver,kidney,spleen, adrenal gland, testis, ovary, seminal vesicle, thyroid gland, hangs down at thymus gland Heart, thymus gland, lung, liver,kidney,spleen, adrenal gland, testis, the ovary, essence of body, the weight of brain tissue and control group B, C group animal Capsule, thyroid gland, hypophysis, brain tissue weight there was no significant difference (P > 0.05) compared to having.

3. 6 months long term toxicities of rat

3.1 experimental methods: EGS antiviral drugs 0.0375,0.075 and 0.15mg/ is injected respectively within SD rat continuous six months Whether kg, observation male and female rat there is any apparent toxicity or death；To the growth of SD rat body weight, whether there is or not obvious for observation Influence, if increase with the extension of test period, food ration, amount of drinking water are had no significant effect, only show it is irregular Increase and decrease fluctuation.Administration mid-term (administration three months), drug withdrawal next day (administration six months), convalescence terminate and (are discontinued one month) inspection Look into, three dosage groups have no significant effect the weight and coefficient of hematology, blood biochemical analysis index and internal organs, if substantially with 0.9% normal saline solution is identical with excipient control group (p > 0.05).Substantially with Histopathology through being examined under naked eyes and mirror It looks into and whether sees obviously because of Pathomorphology and Histological change caused by tested material.

3.2 test result

It is for statistical analysis to data.

(a) (0.9% is raw for the weight and food ration of weight and food ration administration group (three dosage groups) animal and control group Reason brine injections) weight of animal compared that there was no significant difference (P > 0.05) with food ration.

(b) the general health of animal observes the general health and behavioral activity of animal, administration group daily The general health of (three dosage groups) animal and the general health of control group (0.9% normal saline solution) animal Compared to having, there was no significant difference (P > 0.05).

(c) hematology of animal and clinical indices carry out hematology, clinical indices, ophthalmoscopy (method), uridine Analysis checks.All animals will carry out naked eyes and micrography.Hematology and the clinic of administration group (three dosage groups) animal refer to Mark have that there was no significant difference compared with clinical indices with the hematology of control group (0.9% normal saline solution) animal (P > 0.05).

(d) heart, thymus gland, lung, liver,kidney,spleen, adrenal gland, testis, ovary, essence are checked in the postmortem postmortem of animal The weight of capsule, thyroid gland, hypophysis, brain tissue.Not stained slice is carried out to the kidney of blank control group and two high dose group female mices And micrography.The postmortem clinical indices and control group (0.9% normal saline solution) of administration group (three dosage groups) animal The postmortem clinical indices of animal compare heart, the chest for the animal that has that there was no significant difference (P > 0.05) administration group (three dosage groups) (0.9% is raw for gland, lung, liver,kidney,spleen, adrenal gland, testis, ovary, seminal vesicle, thyroid gland, hypophysis, the weight of brain tissue and control group Manage brine injections) animal heart, thymus gland, lung, liver,kidney,spleen, adrenal gland, testis, ovary, seminal vesicle, thyroid gland, hypophysis, brain There was no significant difference (P > 0.05) compared to having for the weight of tissue.

Nine, EGS nucleic acid antiviral drugs Non-clinical Pharmacokinetics research approach

1, research purpose: injection EGS nucleic acid antiviral drugs is investigated in rat and the intracorporal pharmacokinetic situations of macaque, is obtained Basic pharmacokinetic parameter provides reliable reference frame for clinical research and clinical application.

2, oranon: the Non-clinical Pharmacokinetics of injection EGS nucleic acid antiviral drugs are studied according to newest (2007 On October 1, in) it promulgates and in " the drug registration management method " that on October 1st, 2007 implements in biological products registration classification " chemicals Non-clinical Pharmacokinetics investigative technique guideline " ([H] GPT5- of a kind of requirement, in March, 2005 promulgation 1) and the characteristics of biotech drug, is studied.

3. research contents

3.1, the blood concentration-time curve after rat single-dose.

3.2, the Tissue distribution research after rat single-dose.

3.3, blood concentration-time curve after monkey single-dose.

3.4, blood concentration-time curve after monkey multiple dosing.

4, research method, process

4.1, experimental design

4.1.1, the determination of dosage, administration route: according to country to the rule of original new drug Preclinical metabolism and pharmacokinetics study Fixed, the research of this part should carry out in two kinds of animals, and one kind is rodent, and another kind is non-rodent.And The selection of animal should be consistent with the animal of pharmacodynamics and toxicologic study.The selection of dosage should include high, normal, basic three dosage Group, wherein high dose should be effective dose (the quasi- dosage of clinic), low dosage close to the maximum tolerated dose of drug, middle dosage To approach effective dose lower limit.Administration route should be intended that consistent with administration route with pharmacodynamic study and clinic.Therefore, this research Selection carries out in rat and macaque, and dosage is determined according to pharmacodynamic experiment result and acute toxicity testing result, and According to the practical appropriate adjustment in test；The dosage of macaque is converted according to the dosage of rat with body surface area method Come；Administration route is intended that with approach according to pharmacodynamic experiment and clinic and determines.

The middle dosage 100ug/kg(rat used according to EGS pharmacodynamic study), administration mode is intravenous injection.

The dosage of EGS Non-clinical Pharmacokinetics research rat are as follows: high dose 200ug/kg, middle dosage 100ug/ Kg, low dosage 50ug/kg.

The dosage of EGS Non-clinical Pharmacokinetics research monkey are as follows: middle dosage (=rat middle dosage * Rat Standard body Weight * monkey operates with practical 50 ug/kg that press of rat body surface area ratio/standard monkey weight=100*0.2*9.2/4)=46 ug/kg(), Low dosage is 25 ug/kg, and high dose is 100 ug/kg.

4.1.2, administration route: intravenous injection

4.2, experiment content and method

4.2.1, blood concentration-time curve:

1) blood concentration-time curve after rat single-dose: taking healthy rat to be randomly divided into 3 groups (half male and half females), and every group dynamic Object number is no less than 6, gives the EGS of senior middle school's low dosage respectively, in rats with tracer method combination HPLC method measurement EGS Blood concentration-time curve and calculate main pharmacokinetic parameter.Experiment flow is shown in Fig. 9:

2) blood concentration-time curve after macaque (or machin) single-dose: healthy macaque (or machin) is taken to be randomly divided into 4 groups (half male and half female), every group of number of animals is no less than 4, and SC gives senior middle school's low dosage respectively and IV gives the EGS of middle dosage, The blood sampling of preset time point, separated plasma, with the blood medicine of radio-immunity or enzyme-linked immunization measurement EGS in macaque (or machin) Concentration time curve simultaneously calculates main pharmacokinetic parameter.

3) blood concentration-time curve after macaque (or machin) multiple dosing: macaque 4 (half male and half female) are taken to give The EGS of middle dosage, according to Terminal half-life calculated after single-dose and with reference to Pharmacodynamic Data determine dosing interval and to Medicine number of days.Preset time point take a blood sample, separated plasma, with radio-immunity or enzyme-linked immunization measurement EGS macaque (or food crab Monkey) in blood concentration.It needs to provide following result after experiment:

A) the blood concentration-time data and curve and main medicine after each (and each group) animal subject first administration are for power Learn parameter.

B) the 3 Steady state trough concentration data and average value, standard deviation of each (and each group) animal subject.

C) each (and each group) animal subject blood concentration reach stable state after last dose blood concentration-time data and Curve and its average value, standard deviation and curve.

D) compare for the first time with the blood concentration-time curve of last dose and related parameter.

E) each (and each group) Mean steady state concentration of blood drug and standard deviation.

4.2.2, Tissue distribution research

Selection is in rats with the Tissue distribution of tracer method research EGS.Dosage is that middle dosage (contains 5~20 μ Ci/ The 125I- EGS of rat).Method is: taking healthy rat to be randomly divided into 5 groups (half male and half females), every group of animal is no less than 6. According to the result of study of blood concentration-time curve, selecting 5 time points (is distribution 2 time points of phase, inflection point 1 respectively Time point eliminates 2 time points of phase), rat, respectively coring, liver, spleen, lung, kidney, stomach are put to death at corresponding time point after administration The tissue such as enteron aisle, gonad, brain, body fat, skeletal muscle, is divided into two parts, a directly to be measured respectively with gamma counter after being precisely weighed Radiocounting in tissue indicates EGS distribution situation in each tissue with relative accumulation coefficient；Another grouping is knitted middle be added and is fitted It is homogenized after the physiological saline of amount, centrifuging and taking supernatant injects HPLC chromatogram instrument, is separated with gel filtration chromatography column, and distribution is received Collection measures radiocounting with gamma counter, calculates the relative amount percentage of EGS prototype in each tissue.Relative accumulation coefficient Representation formula are as follows:

Relative accumulation coefficient=[radiocounting (CPM)/tissue weight (g)]/[gross activity that every animal is given counts (CPM)/the weight of animals (g)].

EGS relative amount percentage=[total at prototype medicine radiation peak tale (CPM)/all radioactivity peak in tissue Number (CPM)] × 100.

4.3, analysis of experimental results

Using DAS software, the calculating for carrying out pharmacokinetic parameters to MGP plasma concentration is analyzed.

Using 125I Radioactive isotope technology, to injection EGS antiviral drugs in SD rat and macaque (or food crab Monkey) intracorporal pharmacokinetics studied.

SD rat dosage is set as high, medium and low three dosage groups, 200ug/kg, 100ug/kg and 50ug/kg.Macaque (or machin) dosage is set as high, medium and low three dosage groups, 100 ug/kg, 46 ug/kg and 25 ug/kg.3 dosage The ongoing change data of 125I- injection EGS antiviral drugs blood concentration in rat body after injecting are with the practical medicine of 3P87 It is dynamic to learn program processing, select two Room models, weight coefficient to be fitted drug-time curve for 1/C2.The main pharmacokinetic ginseng of 3 groups of rats Number, wherein Cmax and Tmax is indicated with measured value.The T1/2 β of basic, normal, high three dosage is respectively h；AUC0~t is respectively ng·mL-1·h；Cmax is respectively ngmL-1；Tmax is respectively h.

Statistical analysis, test result

(a) after rat single-dose, T1/2 β and Tmax has that there was no significant difference (P > 0.05) between 3 dosage groups；

(b) after monkey single-dose, there was no significant difference between 3 dosage groups (P > 0.05) by T1/2 β and Tmax；

(c) after monkey multiple dosing, there was no significant difference between 3 dosage groups (P > 0.05) by T1/2 β and Tmax；

(d) after rat single-dose, Cmax and AUC0~t increases (P < 0.01) as dosage increases, through Dose standard After change processing, there was no significant difference between 3 dosage groups (P > 0.05) by Cmax,

(e) after monkey single-dose, Cmax and AUC0~t increases (P < 0.01) as dosage increases, through dose normalized After processing, there was no significant difference between 3 dosage groups (P > 0.05) by Cmax,

(f) after monkey multiple dosing, Cmax and AUC0~t increases (P < 0.01) as dosage increases；Through dose normalized After processing, there was no significant difference between 3 dosage groups (P > 0.05) by Cmax,

(g) heart, liver, spleen, lung, kidney, stomach of the Tissue distribution research EGS after rat single-dose after rat single-dose The tissues such as enteron aisle, gonad, brain, body fat, skeletal muscle are distributed between 3 dosage groups that there was no significant difference (P > 0.05).

Claims (3)

1. a kind of synthesis of EGS nucleic acid drug for resisting influenza virus, it is characterized in that it the following steps are included:

Step 1, the target sequence region that popularity common cold virus gene mRNA is cut under EGS guidance RNase P It determines: determining that potential point of contact in target RNA, the high efficiency cutting site of target RNA have following characteristics, i.e.-the 1 of point of contact should be divided with+1 Not Wei pyrimidine (U/C) and guanine (G) ,+8 of point of contact should preferably be uracil (U), therefore, according to 5 '-U/ of sequence general formula CGNNNNNNU-3 ' searches potential point of contact in influenza virus PA, PB1, M1 gene；

Step 2, the design of EGS: the structure of EGS usually by 5 ' combine region sequence, the T stem ring of tRNA and extra loop region sequence and 3 ' combine three parts of region sequence to form, wherein T stem ring and extra loop region sequence are fixed, partial sequences of whole EGS It is selected from people tRNA, 5 ' combined areas and 3 ' combine region sequence then not identical in different EGS, A type stream is found in GeneBank The conserved sequence of Influenza Virus clinical strain's target sequence PA, PB1 and M1 gene, in the fixed situation in potential point of contact, according to this Sequence near point of contact, according to the principle of base pair complementarity, design is directed to the EGS combination region sequence at the point of contact, in design It should be noted that -1 of point of contact cannot be with the base pairing of EGS: should be spaced between sequence target RNA complementary with the two sides EGS combined area 2nt；EGS combination region sequence has 12nt at least to guarantee the specificity of complementary combination as far as possible, designed whole EGS, such as The combined area sequence length of EGS E-PA, E-PB1 and E-M1 are 14-16nt, and 5 ' combined areas and 3 ' combine the length of region sequence It is respectively 7-9 nt；

Step 3, EGS's is artificial synthesized: designed EGS sequence being synthesized, when synthesis adds 2 '-O-methyl chemistry Modification, while the corresponding control EGS (Control EGS, C-EGS) for designed EGS has also been synthesized as control examination It tests, is dissolved EGS lyophilized products with sterile tri-distilled water, and be diluted to 100uM, packing is stored in -20 DEG C, -80 DEG C, spare；

Step 4, the result of synthesis: according to sequence general formula 5'-U/CGNNNNNNU-3', in eight genetic fragments of influenza virus 100 or so potential point of contacts are searched, in addition, there are the conserved motifs of three bases " TTC " in the T ring in EGS, if changed Become conserved motifs, guidance cleavage activity will be substantially reduced, and on the basis of normal EGS, respectively be kept the T environmental protection in each EGS Motif " TTC " all becomes " AAG ", using the control EGS (controlEGS, C-EGS) as corresponding EGS, by In vitro digestion Experiment screening is respectively designated as E-PA, E-PB1, E-M1 to 3 EGS with obvious cleavage activity, they are located at PA, Corresponding EGS, according to the sequence near at these three sites, is designed in the 15 of PB1, M1 gene by 1224,222.

2. the synthesis of EGS nucleic acid drug for resisting influenza virus according to claim 1, it is characterized in that in step 4 The sequence of EGS are as follows:

Number EGS title EGS sequence 1 E-PA 5-GGUUAACAUUGAAGGUGCGGUCUCCGCGCGCAGGUUCAAAUCCUGCUUGUCGCACCA-3 2 E-PB1 5‘-GUAUUGAAGGUGCGGUCUCCGCGCGCAGGUUCAAAUCCUGCGCCCAUCACCA-3‘ 3 E-M1 5‘-GCAAAGCGCGUGCGGUCUCCGCGCGCAGGUUCAAAUCCUGCACGCUGCACCA-3‘

。

3. the synthesis of EGS nucleic acid drug for resisting influenza virus according to claim 1, by three in characterization step four T ring conserved motifs " UUC " in a EGS become " AAG ", simultaneously synthesizing as corresponding control EGS (C33333-EGS) For the EGS of herpes simplex virus type (HSV-1) TK gene, E-TK is as its sequence table of control sequence are as follows:

Number EGS title EGS sequence 1 C-PA 5‘-GGUUAACAUUGAAGGUGCGGUCUCCGCGCGCAGGAAGAAAUCCUGCUUGUCGCACCA-3 2 C-PB1 5‘-GUAUUGAAGGUGCGGUCUCCGCGCGCAGGAAGAAAUCCUGCGCCCAUCACCA-3‘ 3 C-M1 5‘-GCAAAGCGCGUGCGGUCUCCGCGCGCAGGAAGAAAUCCUGCACGCUGCACCA-3‘ 4 E-TK 5-GCUACGUCGGUGCGGUCUCCGCGCGCAGGUUCAAAUCCUGCCGCAGACACCA-3

。