JPH0456804B2 - - Google Patents

Description

[Detailed description of the invention]

TECHNICAL FIELD The present invention relates to antiviral agents. More specifically, the present invention relates to an antiviral agent characterized in that it contains an oligodeoxynucleotide or polydeoxynucleotide equivalent to a partial structure of a DNA sequence that can hybridize to messenger RNA generated during virus replication. It is something. The present inventor has previously discovered that messengers occur during virus multiplication.
Based on this finding, we found that supplying oligodeoxynucleotides or polydeoxynucleotides that are equivalent to a partial structure of a DNA sequence that can hybridize to RNA to the location where the messenger RNA exists inhibits virus proliferation. provided a method for inhibiting virus proliferation (Japanese Patent Application No. 192350/1982), but as a result of repeated experiments in which the above-mentioned oligonucleotides or polydeoxynucleotides were applied to animals infected with viruses, the present invention was developed. We have now provided an antiviral agent. That is, the present invention provides an oligonucleotide equivalent to a partial structure of a DNA sequence that can hybridize to messenger RNA generated during virus proliferation, as a means for carrying out the above-mentioned virus proliferation inhibition method provided by the present inventors. The present invention provides an antiviral agent characterized by containing a deoxynucleotide or a polydeoxynucleotide. The present invention will be explained in detail below. The present inventors first used artificially synthesized RNA to directly add oligodeoxynucleotides equivalent to a partial structure of a DNA sequence that can hybridize to this RNA into a cell-free protein synthesis system. We discovered that proteins that should be synthesized based on RNA are no longer synthesized (see Experimental Example 1 below). In addition, if a similar experiment is performed using an oligodeoxynucleotide with a DNA sequence that cannot hybridize to this RNA, this
It was confirmed that RNA-based protein synthesis occurs. Next, the present inventors discovered that α present in eukaryotic cells
Under physiological conditions using globin messenger RNA and β-globin messenger RNA,
Experiments were conducted using various DANs to determine whether they inhibit the production of α-globin and β-globin. (See Experimental Example 2 below). As a result, by directly adding DNA that can hybridize to α-globin messenger RNA to a cell-free protein synthesis system, production of α-globin is inhibited, and β-globin messenger RNA is inhibited. It has been found that the production of β-globin can be inhibited by directly adding hybridizable DNA to a cell-free protein synthesis system. In addition, α-globin messenger RNA
When using DNA that cannot hybridize to
It was also confirmed that when DNA that cannot hybridize to globin messenger RNA was used, the production of β-globin was not inhibited. Furthermore, in this experimental system, the
It has been found that when the DNA is an oligodeoxynucleotide, a high protein production inhibition rate is exhibited. Based on these experimental results, when we administered oligodeoxynucleotides that form a double strand with messenger RNA, which is formed during the initial stage of infection with herpes simplex virus, to cells infected with herpes simplex virus, we found that syncytia of cells caused by herpes simplex virus was suppressed. It was confirmed that the formation of plaque-like holes (the formation of plaque-like holes in a group of cells on a culture Petri dish due to cell fusion) was significantly inhibited. In the above experiment, calcium chloride was used to increase the uptake of oligodeoxynucleotides into cells, but even when calcium chloride is not used, oligodeoxynucleotides are taken up into cells, and herpes simplex growth occurs in a cell culture system. It was confirmed that virus proliferation was inhibited. (See Experimental Example 3 below) Through these experiments, the growth of herpes simplex virus was effectively inhibited by directly adding DNA capable of hybridizing to messenger RNA of herpes simplex virus to cells infected with herpes simplex virus. However, the present inventor
Furthermore, based on this fact, we applied DNA that can hybridize to messenger RNA of herpes simplex virus to virus-infected mice and conducted a treatment test for viral diseases, and found that it could be used as an antiviral agent. It was confirmed that it is possible. The present invention has been made based on this knowledge. The antiviral agent of the present invention is a DNA that corresponds to the base sequence of messenger RNA derived from a virus.
Because it uses oligodeoxynucleotides or polydeoxynucleotides that are equivalent to a substructure of the sequence, it will not hybridize with messenger RNA derived from other genes, and therefore the use of the antiviral agent of the invention It does not affect protein synthesis by genes. Viruses include DNA viruses (herpesvirus, adenovirus, vaccinia virus, etc.)
It is classified as an RNA virus (influenza virus, rhinovirus, poliovirus, etc.).
RNA viruses include double-stranded viruses and single-stranded viruses, and single-stranded viruses further include positive-stranded viruses and negative-stranded viruses. In any case, viral protein synthesis cannot occur without the presence of positive-strand RNA as messenger RNA. In other words, in double-stranded DNA viruses, plus-strand RNA is produced from minus-strand DNA and becomes messenger RNA; in double-stranded RNA viruses, minus-strand
Plus-strand RNA is made from RNA,
This becomes Metsenjar RNA. Among single-stranded RNA viruses, negative-stranded RNA viruses are
Based on the negative strand RNA, the positive strand
RNA is made and becomes messenger RNA,
In positive-strand RNA viruses, it itself acts as messenger RNA. In the present invention,
Positive-strand RNA as messenger RNA of each virus
By using oligodeoxynucleotides or polydeoxynucleotides equivalent to partial structures of DNA that can hybridize with the virus, an antiviral agent against the target virus is provided. In this specification, the herpes simplex virus, which is a double-stranded DNA virus, will be explained below as an example, and the antiviral agent of the present invention
Regardless of the type of virus, for example, influenza virus, adenovirus, leukemia virus, dengue fever virus, fear virus, hepatitis virus, measles virus, encephalitis virus, parainfluenza virus, New Jersey virus, polio virus, It can be applied to all pathogenic viruses such as rhinovirus, yellow fever virus, and EB virus. Therefore, the present invention can be widely used in the treatment and prevention of various diseases derived from animal and plant viruses, and is therefore extremely useful in various fields. By using the antiviral agent of the present invention, it is possible to stop the proliferation of viruses, regardless of whether they are DNA viruses or RNA viruses, without affecting the non-infected cells of living organisms where the virus exists, and to spread the virus to non-infected cells. infection can be prevented. Regarding the effects of oligodeoxynucleotides or polydeoxynucleotides used in the antiviral agent of the present invention on normal cells, the present inventors have investigated the effect of oligodeoxynucleotides or polydeoxynucleotides used in the antiviral agent of the present invention on single-stranded DNA that can hybridize with messenger RNA of the immediate early gene of herpes simplex virus. When we investigated the effect on fetal hamster kidney cells using a deoxynucleotide having the base sequence, we found that the deoxynucleotide does not affect the survival of normal cells or their proliferation ability. It's clear. Furthermore, during therapeutic experiments on mice infected with herpes simplex virus, no changes were observed in the behavior or appearance of the deoxynucleotide-administered group, which has a single-stranded DNA base sequence that can hybridize with messenger RNA of the immediate early gene. . The oligodeoxynucleotides or polydeoxynucleotides used in the antiviral agent of the present invention are those obtained by decomposing single strands of viral DNA by enzymatic treatment, or
If it has a desired partial structure of the DNA sequence, such as one obtained by synthesizing a partial structure of the sequence,
Its origin does not matter. Examples of methods for preparing oligodeoxynucleotides or polydeoxynucleotides used in the antiviral agent of the present invention include the following methods. That is, one method is to clone the virus's genes and
It is the fragmentation of a single strand of DNA. When cloning viral genes,
In the case of RNA viruses, using reverse transcriptase
Either converted to DNA or based on the viral DNA present in cells infected with the virus. When cloning viral genes,
For example, double-stranded DNA can be obtained using λ phage or a plasmid vector such as pBR322, or single-stranded DNA can be directly obtained using M13 phage or the like. The gene to be cloned may be any part of the nucleic acid chain of the gene contained in the virus, but it is preferable to clone early genes or immediate early genes that are expressed early during proliferation. . In order to reproduce a large amount of cloned DNA, for example, a phage into which the virus gene has been integrated, or E. coli carrying a plasmid, is grown in an appropriate volume of culture solution with antibiotics added as necessary, using a conventional method. After culturing and harvesting, the DNA containing the cloned virus gene is isolated.
During this separation, phenol, phenol-
DNA can be extracted by treatment with an organic solvent such as chloroform. To extract viral DNA from the separated DNA, it is digested with restriction enzymes and then agarose gel electrophoresis, column chromatography, etc. are used. The obtained viral DNA is fragmented as necessary. For this purpose, for example, treatment with a restriction enzyme such as endonuclease or sonication is performed. The chain length of the fragment is usually
It is considered to range from 100 nucleotides to 9 nucleotide positions. When preparing using M13 phage, single-stranded fragments can be obtained directly, but considering the properties of the antiviral agent of the present invention, single-stranded DNA that can form a double strand with the messenger RNA of the viral gene is prepared in advance. Select,
Must be cloned. Since the viral DNA fragment obtained using pBR322 and lambda phage is single-stranded,
For example, after heating at 100°C for 10 minutes, it is rapidly cooled. Since the product contains a single strand of the desired DNA fragment, it can be used as it is in the antiviral agent of the present invention, but this product also contains messenger RNA of the viral gene.
Since there are also single-chain fragments that are not complementary to , it is preferable to remove them using affinity chromatography or the like. Moreover, the oligodeoxynucleotide or polydeoxynucleotide used in the antiviral agent of the present invention can also be produced by organic synthesis. In this case, the base sequence of the oligodeoxynucleotide or polydeoxynucleotide must be a base sequence that can hybridize with the base sequence of the messenger RNA of the viral gene. For example, in the herpes simplex virus, one of the immediate early DNA
It forms a double strand of DNA (5′-ATG, GCG, TCG, GAG…) and minus DNA (3′-TAC, CGC, AGC, CTC…), but among these, the double strand with messenger RNA Select and synthesize the DNA sequence on the TAC side (minus strand DNA) that forms the strand. In addition, for example, in the case of influenza virus, there are hemagglutin genes and NS (Non Structual) genes as influenza virus genes, but in order to inhibit the proliferation of the virus,
It is preferable to stop the expression of the NS gene rather than the hemagglutin gene, which has variations in the DNA base sequence, and it is better to choose a single-stranded DNA base sequence that can hybridize to the messenger RNA of this NS gene. . for example,
Taking the NS1 gene (Lamb et al., Cell 21 475 (1980)) as an example, a single strand that can hybridize to messenger RNA of the NS1 gene
DNA base sequence, e.g. 5′-...ACT, TGA,
CAC・AGT・GTT・GGA・ATC・CAT…−
Select and synthesize a base sequence of an appropriate length in the 3' base sequence. Furthermore, in the case of livestock-related viruses, such as Rous's avian sarcoma virus, gag.
Inhibits the expression of any gene in pol.env.
Here, when inhibiting the expression of the gag gene, for example, considering the base sequence of P19, which is a part of the gag gene, 5'-...AAT, CAC, CTT, TAT,
GAC, GGC, TTC...-Select a base sequence of appropriate length from the -3' base sequence and synthesize it. In this way, in the case of any other virus, we select an appropriate length base sequence in the single-stranded DNA base sequence that can hybridize to the messenger RNA of the virus whose proliferation we want to inhibit. , an oligodeoxynucleotide or a polydeoxynucleotide having the base sequence can be synthesized, and the oligo(or poly)deoxynucleotide can be used. Next, the preparation of oligodeoxynucleotides or polydeoxynucleotides used in the antiviral agent of the present invention will be explained. Specific example 1 Herpes simplex virus was treated with an organic solvent of chloroform-phenol.
Cut the DNA with a restriction enzyme, BamHI, and cut the vector with a restriction enzyme, BamHI in advance.
It was integrated into pBR322 using T4 phage ligase. The obtained pramisud was given to Escherichia coli to form colonies on an agar medium containing ampicillin. Among these colonies, those that hybridize to ^32P -labeled DNA of herpes simplex virus were selected by filter hybridization method, and 2 TSB (Tryptose Soy Broth) containing ampicillin at a concentration of 50 μg/ml was selected.
Cultured in medium. 10 ⁶ to 10 ⁷ E. coli containing the above plasmid were added to 1 ml of this medium, and cultured with shaking in a 20 ml test tube at 37° C. for 15 hours. of culture solution
After culturing until the absorbance at 540 nm reached 0.5, 100 μg/ml of chloramphenicol was added, and the culture was continued for an additional 15 hours. The pellet obtained by centrifuging the E. coli culture solution thus obtained contains 25% sucrose.
Add 50mM Tris-HCl (PH=8.0) and
Leave it at below ℃ for 5 minutes and add 4 ml of Tri-ton-X100.
The viscous liquid produced by adding
Centrifuged for minutes. Add 8g of CsCl to 8ml of this supernatant and
After adding 1 ml of Ethidium Bromide (5 μg/ml) and further centrifuging at 100,000 G for 48 hours, the resulting crude plasmid fraction was collected, mixed with twice the amount of isopropyl alcohol, and the supernatant was removed. Dialysis was performed overnight in 0.1M Tris 10mM EDTA (PH=8.0). After concentrating the dialysed fluid and taking an amount equivalent to 200 μg of DNA, adding an excess amount of restriction enzyme (BamHI) and reacting, using agarose electrophoresis,
The DNA of herpes simplex virus was isolated. This DNA was then partially degraded with DNAase, heated at 100℃ for 10 minutes, and then rapidly cooled to reduce the chain length to 9-100.
The oligodeoxynucleotide or polydeoxynucleotide of the position is obtained. Example 2 M13 phage replicative duplex for cloning
DNA was cut with BamHI. Next, the DNA of the herpes simplex virus isolated in Example 1,
This cleaved M13 phage replicative duplex
DNA was ligated using T4 phage ligase.
The ligated DNA was placed into lac(-) E. coli in the presence of CaCl ₂ in a conventional manner, and phage plaques were made in the presence of X-gal (indicator). M13 phages containing herpes simplex virus DNA formed phage plaques that did not exhibit blue color after 24 hours. Herpes simplex virus labeled with ^32P
Plaques that hybridized with the positive strand of DNA (strand length of 20 deoxynucleotides or more) were collected. This indicates that the phage plaque, which does not show blue color, contains the double strands of the herpes simplex virus.
This is because only one side of the DNA is contained. This M13 phage was treated with phenol chloroform to isolate DNA. The DNA obtained above contains herpes simplex virus single-stranded DNA, but it was further purified to clarify the M13 phage contained therein.
The DNA may be removed or partially degraded with DNAase to obtain oligodeoxynucleotides or polydeoxynucleotides having a chain length of 9 to 100 positions. Specific example 3 Using a DNA synthesizer (manufactured by Applied Biosystem), oligodeoxynucleotides (3' −…
TAC・CGC・AGC・CTC・TTG・TTC・
20 deoxynucleotides (3'-CGC, AGC, CTC, TTG, TTC, GTC, GCG...-5')
GTC・GC-5′) was synthesized and purified. Hereinafter, this 20 deoxynucleotide will be abbreviated as 20mer.
This 20mer sequence was confirmed by the Maxam-Gilbert method. Specific Example 4 Using a DNA synthesizer (manufactured by Applied Biosystem), the oligodeoxynucleotides that form a double strand with the minus DNA of the immediate early DNA of the herpes simplex virus and the plus DNA 20 deoxynucleotides (3′-GCA, CCC, GGG,
ACC・TTT・ACC・GC-5′) was synthesized and purified. Below, these 20 deoxynucleotides are 20B
It is abbreviated as This 20B sequence was confirmed by the Maxam-Gilbert method. The antiviral agent of the present invention can be prepared by adding the above-mentioned oligodeoxynucleotides or polydeoxynucleotides, as appropriate, using appropriate solvents, excipients, adjuvants, etc. that are normally used in pharmaceutical formulations, in a manner that is commonly used in pharmaceutical preparations. It is prepared as a liquid, injection, suppository, etc. according to the law. For formulation, desired oligodeoxynucleotides or polydeoxynucleotides may be used alone or in appropriate combinations, and other pharmaceutically active ingredients may also be blended. In these combinations and formulations, the components to be combined or formulated must not inhibit the antiviral action of the oligodeoxynucleotides or polydeoxynucleotides described above. The above-mentioned oligodeoxynucleotides or polydeoxynucleotides can be mixed with a suitable base that is inactive against them to form external preparations such as creams, ointments, and poultices. When preparing a solution or injection, distilled water for injection, physiological saline, aqueous dextrose solution, vegetable oil for injection, propylene glycol, polyethylene glycol, etc. can generally be used. Furthermore, if necessary, isotonizing agents, solubilizing agents,
Stabilizers, preservatives, soothing agents, etc. may also be added. Moreover, in the case of this type of dosage form, it is desirable to dissolve it in a sterile injection medium. Various bases and additives such as those mentioned above used in the preparation of formulations,
It goes without saying that the adjuvant and the like must not inhibit the antiviral action of the oligodeoxynucleotide or polydeoxynucleotide described above. The antiviral agent of the present invention is applied directly to the affected area of a living body infected with a virus, or administered intravascularly, so that the oligodeoxynucleotide or polydeoxynucleotide eventually reaches the affected area. Adapt it to the living body so that it can be obtained. Examples of experiments to confirm the effectiveness of the antiviral agent of the present invention using mice are listed below. Example 1-(1) HSV-1 (F
(1×10 ⁴ PFU) was injected intraperitoneally into the peritoneal cavity with 200μ of 20mer MEM solutions at various concentrations.
(contains 1% penicillin and streptomycin and 2mM glutamine).
1 (F strain) was administered daily for 3 days from the day after injection,
The mortality rate was examined on the 7th day after HSV-1 (strain F) injection. As shown in Figure 1, in the 20mer administration group, the mortality rate due to HSV-1 was reduced in a concentration-dependent manner. The mortality rate was expressed as the mortality rate in the 20mer non-administration group as 100%. Example 1-(2) Inject HSV-1 (F
HSV-1 (F) (1×10 ⁴ PFU)
Inject 30μ (1×10 ⁴ PFU) of MEM solution of M.I.
200μ of MEM solution (containing 1% penicillin and streptomycin and 2mM glutamine) was administered daily for 6 days starting the day after HSV-1 (strain F) injection, and 64 to 68 hours after injection of HSV-1 (strain F). The eye mortality rate was investigated. The results are shown in Table 1.

[Table] As shown in Table 1, the mortality rate in the 20mer non-administration group was 5/19, whereas no death was observed in the 20mer administration group. Example 1-(3) HSV-1 (F
(5×10 ⁴ PFU) was injected,
Mortality rates were examined 64 to 68 hours and 71 to 78 hours after HSV-1 (strain F) injection. The results are shown in Table 2.

[Table] As shown in Table 2, the mortality rate was 3/10 (64 to 68 hours) and 3/10 (71 to 78 hours) in the 20mer non-administration group, whereas the mortality rate in the 20mer administration group was clearly was observed to be decreasing. Example 1-(4) HSV-1 (F
(5×10 ⁴ PFU) was injected into the ventricles of these mice, and various concentrations of 20mer MEM were injected into the ventricles of these mice.
30μ of solution (contains 1% penicillin and streptomycin and 2mM glutamine)
was administered every day for 2 days from the day after HSV-1 (F strain) injection, and 64 to 68 hours after HSV-1 (F strain) injection, 71
Mortality was examined at ~78 hours and between 88 and 92 hours. The results are shown in Table 3.

[Table] As shown in Table 3, it was observed that the mortality rate in the 20mer administration group was clearly reduced up to 88-92 hours compared to the mortality rate in the 20mer non-administration group. Example 1-(5) Inject 1.12μ into the peritoneal cavity of a 3-week-old BALB/C mouse.
HSV-1 in 200 μg/ml 20mer MEM solution
(F strain) containing (1 × 10 ⁶ PFU) was injected,
This mouse received various concentrations of 20mer intraperitoneally.
200μ of MEM solution (containing 1% penicillin and streptomycin and 2mM glutamine) was administered daily for 3 days from 3 hours and 2 hours before HSV-1 (strain F) injection and the next day.
The mortality rate was examined at 211 hours and 259 hours after HSV-1 (strain F) injection. The results are shown in Table 4.

[Table] As shown in Table 4, the mortality rate was 1/10 (211 hours) and 2/10 (259 hours) in the 20mer non-administration group, whereas no death was observed in the 20mer administration group. Example 2: A 3-week-old child was injured five times with a sharp needle under anesthesia.
Apply 30μ of MEM solution to the cornea of BALB/C mice.
(containing 1% penicillin and streptomycin and 2mM glutamine and 5% calf serum) plus HSV-1 (strain F)
10 ⁶ PFU/ml) was added dropwise. The above mice were divided into the following four groups and their behavior was observed. A: 100μ of a MEM solution containing 22.4μg/ml of 20mer was instilled into the cornea of this mouse 2 hours before HSV-1 (strain F) and three times every other day starting from the next day. 1 day before instillation, 5
A group in which 30μ of cortisone acetate solution (2mg/Kg) was intramuscularly injected 3 times before and every other day starting from the next day. B: A group in which 10μ of a MEM solution containing 22.4μg/ml of 20mer was dropped onto the cornea of the mouse three times, 2 hours before HSV-1 (strain F) was dropped, and every other day starting from the next day. C Apply 10μ of MEM solution to the cornea of this mouse.
HSV-1 (F strain) was instilled into the cornea 3 times every other day, starting from 2 hours before and the next day.
Cortisone acetate solution (2 mg/Kg) 30μ administered every other day starting from 1 day before instillation, 5 hours before instillation, and the next day.
The group received intramuscular injection. D Apply 10μ of MEM solution to the cornea of this mouse.
-1 (F strain) A group in which the drops were applied to the cornea three times every other day starting from 2 hours before and the next day. When we observed the mice in each group on the 4th, 7th, and 12th day, focusing on the condition of the eyes and the rash seen around the eyes, we found that group A, which is the group to which 20mer was administered; In group B, groups C and D are non-administered groups.
Compared to the group, there was clearly less rash and the condition of the eyes was good. At this time, regarding the effect of 20mer administration,
No effect of cortisone administration was observed. From the above Examples 1 and 2, it is clear that the antiviral agent of the present invention is highly useful because it suppresses the proliferation of the virus in affected animals, thereby reducing the mortality rate and alleviating the symptoms. It turns out that it is expensive. Examples of formulations of the antiviral agent of the present invention are listed below. Formulation Example 1 A product similar to the 20mer obtained in Specific Example 3 was synthesized by the liquid phase method, and 2g of the 20mer was added with an isotonic MEM solution according to the usual method for manufacturing injections.
It is sealed in an ampoule and used as an injection. This injection contains 2 mg of 20mer in 1 ml. Inject 1 to 5 ml of this injection at a time, depending on the symptoms, as close as possible to the infected area. Formulation Example 2 A product similar to 20B obtained in Example 4 was synthesized by a liquid phase method, and an isotonic MEM solution was added to 10 g of the 20B to prepare a 1% eye drop. This eye drop is 2 to 3 drops at a time, depending on the symptoms.
Apply eye drops several times a day. Formulation Example 3 A product similar to the 20mer obtained in Specific Example 3 was synthesized by the liquid phase method, and 1 g of the 20mer was polished to a fine powder. 999 g of purified cacao butter was added to this and kneaded on a 60°C water bath. Shape into suppositories weighing 2g each. Each suppository contains 2mg of 20mer and is used depending on the symptoms. Next, the safety of the antiviral agent of the present invention will be explained by showing test examples. Test Example 1 100 μg/Kg of 20mer was administered to BALB/C mice.
No deaths were observed when administered ip. Test Example 2 In the aforementioned effect confirmation experiment, Example 2, HSV-1
In an experiment conducted without using , the other things were the same,
No difference was observed between the 20mer non-administration group and the 20mer administration group. As shown here, it is clear that the antiviral agents of the present invention are safe at effective doses of the oligodeoxynucleotides or polyoxynucleotides described above. Examples of experiments conducted in connection with the present invention are listed below. Experimental Example 1 Inhibitory effect of polyadenylic acid and deoxypolyadenylic acid on polyphenylalanine synthesis reaction using polyuridylic acid as messenger RNA Polyuridylic acid (14 μg), ¹⁴ C-phenylalanine (10 ⁵ cpm, 300 μCi/μmole), Ni- erenberg
and Mathaei's method (Proc.Nat.Acad.Sci.47,
1588 (1961)),
13mM Magnesium Acetate, 1mM ATP, 1mM
A reaction solution system was prepared by dissolving creatine phosphate and creatine phosphokinase (1 μg) in 10 mM Tris-HCl (PH7.5). The inhibitors listed in Table 1 were added to this reaction solution system to make it 40μ, and the mixture was reacted at 37°C for 30 minutes in a 1.5ml Etzpendorf tube, and the amount of polyphenylalanine in the solution obtained by the reaction was reduced to 10%.
The amount of radioprotein insoluble in trichloroacetic acid at 95°C was measured using a liquid scintillation counter. The degree of inhibition was calculated by comparing the radioactivity of polyphenylalanine when the inhibitor was not added and the radioactivity when the inhibitor was added. The results are shown in Table 5.

[Table] As shown in Table 5, polyuridylic acid (RNA)
On the other hand, polydeoxyadenylic acid (DNA) corresponding to the base sequence showed a remarkable activity of inhibiting the synthesis of polyphenylalanine. In this experiment, DNA strands showed strong inhibitory activity up to oligodeoxyadenylates (oligodeoxynucleotides) of 9 or more nucleotides. Experimental Example 2 Specific inhibition effect of globin single-stranded DNA on globin synthesis in the reticulocyte system of rabbits.
This is an example of an experiment in which it was confirmed that protein synthesis can be inhibited under physiological conditions using oligodeoxynucleotides or polydeoxynucleotides that hybridize to RNA. 4.2mM calcium phosphate, 2mM dimethylthiothreitol (DTT), 0.08mM amino acids (excluding methionine), 6mM potassium acetate, 8mM magnesium acetate, 4.5μg spermidine, ^35S -methionine (0.1μCi), and Ja- ckson and Pelham et al. Cell-free reticulocyte protein synthesis system 10 μ, prepared by the method of (Eur. J. Biochem. 67.247-256 (1976))
α and β globin messenger RNA 75 μg each
was dissolved in 21mMHEPES and the inhibitors listed in Table 6 were added to make a total volume of 30μ, followed by reaction at 30°C for 30 minutes in a 1.5ml Etzpendorf tube. The reaction products in the solution obtained by the above reaction were separated into α globin, β
The protein was separated into globin and other proteins, and the radioactivity of each fraction was measured using radioautography. The degree of inhibition was calculated by comparing with the radioactivity when no inhibitor was added. The results are shown in Table 6.

[Table] As shown in Table 6, the genome of α-globin
DNA specifically inhibits only the synthesis of α-globin, and genomic DNA of β-globin specifically inhibits only the synthesis of β-globin. Calf thymus DNA showed almost no inhibitory activity. This indicates that the presence of DNA corresponding to the base sequence of messenger RNA in eukaryotic cells specifically inhibits protein synthesis based on the messenger RNA. moreover,
Since the 15-oligodeoxynucleotide corresponding to the N-terminal amino acid of α-globin inhibits only the synthesis of α-globin, and the 15-oligodeoxynucleotide of M13phage has little effect on the synthesis of α-globin and β-globin, it was found that It was found that even when the DNA strand corresponding to the RNA base sequence was shortened to 15 nucleotides, its inhibitory activity and specificity were not lost. Experimental Example 3 Permeability of 20mer into fetal hamster kidney cells (BHKcell) This experimental example was an experiment to confirm the permeability of single-stranded oligonucleotide into cells. Hamster fetal kidney cells (BHKcell, 4.4×
10 ⁵ cells) were cultured for 3 hours in the presence or absence of HSV-1 (7.6×10 ⁴ PFU/ml). 17.66 pmol of P-labeled 20mer for these cells
were contacted for various times. Remove the solvent and wash the cells twice with 0.3 ml of PBS.
Furthermore, 0.1M NaCl, 33μM for 30 minutes at 36℃
ZnCl ₂ , 33.3mM containing 3360units nuclease S1
After standing in a solution of sodium acetate buffer (PH4.5), it was washed twice with the above sodium acetate buffer without nuclease S1, and 0.3ml of 10mM
EDTA, 0.6% sodium dodecyl sulfate and
Cells were lysed with 10mM Tris buffer (PH7.5). Here, the TCA insolubility in this solution and
When the amount of 20mer sensitive to nuclease S1 was measured, 4.68 x ¹⁰³ 20mers per cell in 8 hours.
It was found that it was taken in. Experimental example 4 Effect of blending oligodeoxynucleotides (a) Microplate (manufactured by Linbro, hole inner diameter
Place 1.5 x 10 ⁵ fetal hamster kidney cells (BHKcell) per hole in a 1.5 cm) and culture at 36°C for 47 to 48 hours in a 5% CO ₂ incubator. 160 μ of Tux virus (HSV-1) (7.6×10 ⁴ PFU/ml) was added, and infection was carried out at 36° C. for 3 hours. Inject 0.3 ml of various amounts of 20mer or a mixture of 20mer and 20B (224:1) into the infected cells in a solvent.
( _CaCl2 concentration 4.55mM) for 8 hours. (b) Separately, a culture operation was performed in the same manner as in (a) above, except that 20mer or a mixture of 20mer and 20B was not used (virus control). The cultured cells (a) above were fixed, stained, and the number of syncytia was counted. Use this result to determine the number of syncytia in the virus control (b).
It is shown in Figure 2 as 100%. As shown in Figure 2, when a mixture of 20mer and 20B was used, it was found that the virus growth inhibition effect was stronger than when 2mer was used alone. From this, it was confirmed that the inhibitory effect may be enhanced when different base sequence parts of the base sequences constituting the minus-strand DNA of the herpes simplex virus are used together.

[Brief explanation of the drawing]

FIG. 1 is a graph showing the results of an example (example 1-(1) described above) for confirming the effect of the antiviral agent of the present invention. The vertical axis of the graph represents the mortality rate of the 20mer administered group relative to the mortality rate of the 20mer non-administered group, and the horizontal axis represents the dose of 20mer (μg/mouse). Figure 2 shows the case of using 20mer alone and the case of using 20B in Experimental Example 4 of this specification.
This is a graph to compare the virus growth inhibition effect when using a mixture of
The percentage of syncytium number in the group using 20mer or a mixture of 20mer and 20B is taken, and the amount of 20mer used or the amount of a mixture of 20mer and 20B used is plotted on the horizontal axis.

Claims (1)

[Claims] 1 DNA that can hybridize to messenger RNA generated during virus replication
An antiviral agent characterized in that it contains oligodeoxynucleotides or polydeoxynucleotides that are equivalent to a partial structure of the sequence.