KR101293363B1 - A Composition for Preventing Bird Flu Comprising Interferon Receptor gene - Google Patents

Abstract

The present invention provides a composition for preventing avian influenza comprising a polynucleotide encoding an interferon receptor capable of inhibiting intracellular proliferation of avian influenza virus or a vector comprising the polynucleotide, and preventing avian influenza comprising the polynucleotide or the vector. A method for preventing avian influenza comprising administering a DNA vaccine and a polynucleotide, vector, composition or DNA vaccine to a subject. Using the method of the present invention, while suppressing the proliferation of avian influenza virus, can eliminate the side effects caused by inteferon, it will be widely used for the prevention of avian influenza caused by avian influenza virus.

Description

A composition for Preventing Bird Flu Comprising Interferon Receptor gene}

The present invention relates to a composition for preventing avian influenza comprising an interferon receptor gene, and more specifically, the present invention includes a polynucleotide or the polynucleotide encoding an interferon receptor capable of inhibiting intracellular proliferation of avian influenza virus. Avian influenza preventive composition comprising a vector, the polynucleotide or avian influenza DNA vaccine comprising a vector and preventing the bird flu comprising administering the polynucleotide, vector, composition or DNA vaccine to a subject It is about a method.

Influenza virus is an RNA virus belonging to the family Orthomyxoviridae, and serotypes are divided into three types: A, B or C. Among them, type A is known to be infected by humans, horses, pigs, other mammals, and various kinds of poultry and wild birds, also known as Avian Influenza (Selmons et al., Avian Dis., 18 (1) : 119-124 (1974); Turek R et al., Acta Virol., 27 (6): 523-527 (1983); Webster RG et al., Microbiol Rev., 56 (1): 152-179 ( 1992). The avian influenza is spread quickly and pathogenic, and is infected with various kinds of birds, such as chickens, turkeys, wild birds, mainly acute viral infectious diseases that damage chickens and turkeys. In particular, duck is a disease that does not show any clinical symptoms even if it is infected. Among them, Highly Pathogenic Avian Influenza (HPAI) is required to be reported by the International Water Agency (OIE). Since the first case of human infection caused by avian influenza virus has been reported, it is a disease that is very important in public health.

Serotypes of avian influenza viruses are classified according to the two types of proteins on the surface of the virus, biological glutinin (Hemagglutinin (HA) and neuraminidase (NA), and 144 types (HA protein 16) Species and 9 NA proteins). The HA is responsible for attaching the virus to somatic cells, and NA is known to allow the virus to penetrate into cells (Alexander DJ, Vet. Microbiol., 74 (1-2): 3-13 (2000)). A global epidemiological study of influenza infection in wild birds confirms that all existing 16 HA and 9 NA influenza viruses are infected in wild birds (Selmons et al., Avian Dis. , 18 (1): 119-124 (1974)). Avian influenza virus, classified as type A, is a common infectious disease virus, a non-pathogenic avian influenza that causes mild respiratory symptoms when infected with chickens, and is low pathogenic, causing death and scattering around 1-30%. Low pathogenic avian influenza (LPAI) and high pathogenic avian influenza (HPAI) with a high mortality rate of more than 95% and also called "bird flu" (Alexander DJ, Vet. Microbiol., 74 (1-2): 3-13 (2000)).

High pathogenic avian influenza (HPAI) has been confirmed worldwide since 1980, including the United States, Australia, Pakistan or Hong Kong. In particular, in December 2003, serotype A / H5N1 highly pathogenic avian influenza occurred in Southeast Asia and the Far East Asia, including Vietnam, Japan or Thailand, in 2004. In particular, the highly pathogenic avian influenza in Vietnam and Thailand has been confirmed to be a mutant avian influenza (mutant A / H5N1 HPAI) virus that can infect a human body when contacted with an infected algae, unlike the traditional highly pathogenic avian influenza which does not infect the human body. Song CS et al., Korean J. Poult. Sci., 31 (2): 129-136 (2004).

Influenza viruses infect the respiratory system, causing systemic symptoms, periodic changes in appearance, and not killing the host, but moving to another host before the host dies. Although the vaccine has been developed against it, The magazine is not available yet, and the fundamental virus treatment is not done yet. Meanwhile, amantadine and rimantadine are substances that inhibit the function of M2 ion channel protein of influenza virus and are representative antiviral agents that inhibit the growth of influenza virus in vivo. However, these two antiviral agents have been found to have the disadvantage that the emergence of a mutant virus that does not affect the ion channel function of the influenza virus M2 protein is very easy. Zanamivir and oseltamivir (Tamiflu), which are developed to compensate for these disadvantages, are substances that inhibit the function of the neuraminidase protein of influenza virus and inhibit the proliferation of influenza virus in vivo. Representative antiviral agents. However, zanamivir has the disadvantage of inhalation and intravenous administration, while oseltamivir can be administered orally, but it has been pointed out as a disadvantage due to side effects such as recent reports of the emergence of resistant virus and vomiting and dizziness upon oral administration (Ward P et. al., J. antimicrob. Chemother., 55: 15-121 (2005)).

On the other hand, the Mx protein is a monomeric GTPase, which inhibits viral replication, and thus is generally known to have an antiviral effect (Samuel, Virology 183: 1-11 (1991)). This antiviral effect of Mx has been demonstrated in negative strand RNA viruses (eg, orthomyxovirus), but the Mx protein itself is expressed in all cells with type 1 interferon (IFN) receptors. Genes encoding the Mx protein are known to induce their expression in response to elevated levels of interferon (IFN) in the body induced in response to viral infection (Bazzigher, L., et al., Virology, 186: 154-160, 1992). Since expression of Mx protein is dependent on the level of IFN in the body, the Mx protein is expressed equally in acute and chronic viral infections that increase the level of IFN in the body (Fernandez, M., et al., J. Infectious Diseases, 180: 262-267, 1999).

Under this background, the present inventors were expected to inhibit the proliferation of avian influenza virus when the over-expression of interferon intracellularly, the side effect of inducing apoptosis (apoptosis) when the interferon is present at a certain level in the cell Therefore, as a result of intensive studies on whether interferon receptors can be used instead of interferon, cells expressing interferon receptors are resistant to avian influenza virus infection, thereby preventing cell death by avian influenza virus infection. In addition, it was confirmed that it is possible to inhibit the proliferation of avian influenza virus in infected cells and completed the present invention.

It is an object of the present invention to provide a composition for preventing avian influenza comprising an interferon receptor gene capable of inhibiting the proliferation of avian influenza virus in a cell.

Another object of the present invention is to provide a DNA vaccine comprising an interferon receptor gene capable of inhibiting the proliferation of avian influenza virus in a cell.

Still another object of the present invention is to provide a method for preventing avian influenza by administering the interferon receptor gene, the composition or the DNA vaccine to an individual.

In order to achieve the above object, according to an embodiment of the present invention, the present invention comprises a polynucleotide encoding the receptor of the interferon or a vector comprising the polynucleotide as an active ingredient, comprising a pharmaceutically acceptable carrier Provided is a pharmaceutical composition for preventing bird flu.

The inventors note that the expression of various antiviral proteins such as Mx, RNase L, PKR, OAS, etc., which are known to exhibit antiviral effects, are induced by interferon. Although resistance to infection may be enhanced, the interferon has a side effect of inducing apoptosis when present in a cell at a certain level or higher, even if overexpressing the interferon receptor instead of the interferon is the same antiviral We wanted to see if it could work.

In order to confirm this, the inventors confirmed the sequence information of the chicken interferon receptor (IFNAR) gene based on the sequence information registered on the gene bank through NCBI (http://www.ncbi.nlm.nih.gov/). (ChIFN-α: X92476 / chIFN-β: NM 001024836), and based on this, the recombinant IFNAR gene (IFNAR1 gene encoding the IFN-α receptor (SEQ ID NO: 1) and IFNAR2 gene encoding the IFN-β receptor (SEQ ID NO: 2)) was obtained. As a result of verifying the antiviral activity of the obtained recombinant IFNAR gene, the cells into which the recombinant IFNAR gene was introduced inhibited nearly 35% of the avian influenza virus H9N2 proliferation, and when the recombinant IFNAR gene and IFN were simultaneously treated It was confirmed that the inhibitory effect was further increased by 10 to 20%, and this effect was shown not only to avian influenza virus but also to paramyxoviruses causing diseases in humans such as NDV. Since it can show resistance to most viral infections, it can be seen that it exhibits a universal preventive effect against viral diseases.

According to another embodiment of the present invention, the present invention provides a polynucleotide encoding a receptor of an interferon or a vector comprising the polynucleotide as an active ingredient, and a DNA vaccine for avian flu prevention comprising a pharmaceutically acceptable carrier. to provide.

As used herein, the term "DNA vaccine" refers to a vaccine in the form of a plasmid that encodes a specific antigen and contains a gene as a vaccine different from the existing protein vaccine. The DNA vaccine is introduced into various cells including dendritic cells in the vaccinated body upon immunization, inducing transcription of antigen genes and production of antigens, and presenting antigens to B cells and T cells to induce humoral and cellular immune responses. Therefore, it is possible to induce continuous antigen expression in the body without major adverse effects on the human body, more effective in inducing cellular immune response, easy to manufacture and produce, and stable at room temperature, so that stable storage and distribution are easy. There is this.

According to another embodiment of the invention, the invention provides a polynucleotide encoding an interferon receptor, a vector comprising said polynucleotide, said polynucleotide or composition comprising said vector, or a DNA vaccine comprising said polynucleotide or vector. It provides a method for preventing avian flu, comprising the step of administering.

As used herein, the term "prevention" refers to any action that inhibits or delays the onset of a disease caused by avian influenza virus infection by administration of a composition comprising the gene encoding the interferon receptor.

As used herein, the term "individual" refers to poultry such as chickens and ducks having diseases caused by direct and indirect causes due to avian influenza virus infection. By administering the gene, composition or DNA vaccine of the present invention comprising the nucleic acid to the subject, it is possible to effectively prevent diseases such as bird flu caused by the avian influenza virus infection described above. The composition of the present invention may be administered in parallel with an existing avian influenza virus infection disease prevention agent.

As used herein, the term "administration" means introducing a predetermined substance into an individual in any suitable manner, and the route of administration of the composition of the present invention is oral or parenteral via any general route as long as it can reach the target tissue. May be administered. In addition, the composition may be administered by any device capable of transferring the active agent to the target cell. Preferred modes of administration are subcutaneous, intradermal, intramuscular, intravenous, and the like.

The composition of the present invention is administered in a pharmaceutically effective amount. As used herein, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and an effective dose level refers to the sex, age, severity, activity of the drug of the individual. , Drug sensitivity, time of administration, route of administration and rate of release, duration of treatment, factors including concurrently used drugs, and other factors well known in the medical arts. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. It may be single or multiple doses. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without adverse effect, and can be easily determined by those skilled in the art. The effective amount may vary depending on the route of treatment, the use of excipients and the possibility of use with other agents, as will be appreciated by those skilled in the art, but in general the effective dose of the composition of the present invention is 0.01 to 0.2 mg at a time per adult / Kg is preferred and can be administered one or more times at intervals of two to four weeks.

The composition or DNA vaccine of the present invention may be administered alone with the gene encoding the interferon receptor or co-administered with other adjuvants or cytokines known in the art. Cytokines that can stimulate an immune response that can be coadministered include, for example, granulocyte-macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (GCSF), IL-1, IL-2, IL-3, IL -4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-15, TNF-α, INF-γ, Flt3 ligand, and the like.

The compositions or DNA vaccines of the present invention utilize agents that promote the influx of genes encoding interferon receptors, such as liposomes, or with lipophilic carriers of one of many sterols, including cholesterol, cholate and deoxycholic acid. It can also mix | blend. It may also be conjugated to a peptide that is taken up by the cell. Examples of useful peptides include peptide hormones, antigens or antibodies and peptide toxins.

The composition or DNA vaccine of the present invention may be administered with a pharmaceutically acceptable carrier, and upon oral administration, a binder, a suspending agent, a disintegrant, an excipient, a solubilizer, a dispersant, a stabilizer, a suspending agent, a pigment, a perfume, etc. In the case of topical administration, bases, excipients, lubricants, preservatives and the like can be used. Injections include non-aqueous solvents such as aqueous solvents such as physiological saline solution and ring gel solution, vegetable oils, higher fatty acid esters (e.g., oleic acid ethyl), and alcohols (e.g., ethanol, benzyl alcohol, propylene glycol, glycerin, etc.). Stabilizers (e.g. ascorbic acid, sodium bisulfite, sodium pyrosulfite, BHA, tocopherol, EDTA, etc.), emulsifiers, buffers for pH control, to prevent microbial development Preservatives such as mercury nitrate, chimerosal, benzalkonium chloride, phenol, cresol, benzyl alcohol, and the like. The pharmaceutical compositions of the present invention can be prepared in a variety of formulations. For example, in the case of oral administration, it may be prepared in the form of tablets, troches, capsules, elesir, suspensions, syrups, wafers, etc., and in the case of injections, may be prepared in unit dosage ampoules or multiple dosage forms.

Using the method of the present invention, while suppressing the proliferation of avian influenza virus, can eliminate the side effects caused by inteferon, it will be widely used for the prevention of avian influenza caused by avian influenza virus.

1 is a schematic diagram showing the structure of IFNAR1 and 2 genes.
Figure 2 is a schematic diagram showing the results of analyzing the common base sequence of different species of human, mouse and chicken for efficient RT-PCR.
Figure 3 is an electrophoresis picture showing the results of the extraction of IFNAR1 and 2 genes.
Figure 4 is an electrophoresis picture showing the result of confirming the size of the insert inserted in the vector.
5 shows the results of confirming the nucleotide sequences of fragments 1 and 2 of chIFNAR1.
6 shows the results of confirming the nucleotide sequences of fragments 1 and 2 of chIFNAR2.
7 is a schematic diagram showing the genetic background of chIFNAR1.
8 is a schematic diagram showing the genetic background of chIFNAR2.
Figure 9 is an electrophoretic picture showing the results of confirming the size of the insert inserted in the vector.
10 shows the results of confirming the nucleotide sequences of chIFNAR1 and 2.
FIG. 11 is a confocal micrograph showing the results obtained by introducing plasmid vectors of pcDNA chIFAR1 and 2 into HeLa cells and confirmed by immunofluorescence analysis.
12 is a photograph showing the results confirmed by Western blot analysis after introducing the plasmid vectors of pcDNA chIFNAR1 and 2 into HEK-293T cells.
FIG. 13 is a photograph and a graph showing the results of confirming that overexpression of IFNAR1 and 2 inhibits the proliferation of the virus and amplifies the antiviral effect by chIFN.
14 is a histogram and graph showing the results of quantitative analysis of the degree of inhibition of virus proliferation when overexpressing chIFNAR1 and 2 in chicken cells.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example  1: of chicken IFNAR1 ,2 gene Cloning

Example  1-1: primer  design

Sequence information of chicken interferon receptors (IFNAR1 and IFNAR2) was confirmed based on sequence information registered on GenBank through NCBI (http://www.ncbi.nlm.nih.gov/) (chIFNAR1: AY744159 / chIFNAR2: AY538711). The primer set was designed to clone the genes of IFNAR1 and 2 of the chicken in the mammalian expression vector pcDNA 3.1a vector to overexpress the chicken cells. At this time, RNA was extracted from chicken cells and cloned by cDNA synthesis method through RT-PCR. For this, primers were designed by dividing the sequence of each IFNAR1 and 2 genes into fragments 1 and 2 (FIG. 1). On the other hand, for efficient RT-PCR, a sense primer of each gene was designed by analyzing common nucleotide sequences of different species of human, mouse and chicken (FIG. 2).

chIFNAR1 fragment 1

sense: 5'-ggatccgccgccaccatgtatgcttgtgcttctgggc-3 '(SEQ ID NO: 3)

antisense: 5'-gaacaggcctcctttatcc-3 '(SEQ ID NO: 4)

chIFNAR1 fragment 2

sense: 5'-ggataaaggaggcctgttc-3 '(SEQ ID NO: 5)

antisense: 5'-ctcgagtagtatgtcatttcctagtgtt-3 '(SEQ ID NO: 6)

chIFNAR2 fragment 1

sense: 5'-ggtaccgccgccaccatggaatcactgatgattggcccacttcggtttactcagc-3 '(SEQ ID NO: 7)

antisense: 5'-ccaacggatccttctgcc-3 '(SEQ ID NO: 8)

chIFNAR2 fragment 2

sense: 5'-ggcagaaggatccgttgg-3 '(SEQ ID NO: 9)

antisense: 5'-ctcgagtcttcgtacgtattcactttt-3 '(SEQ ID NO: 10)

Example  1-2: PCR

10-day-old fertilized eggs were received from the National Institute of Animal Science, and primary trachea epithelial cells (TECs) were separated using the Percoll gradient method and cultured at 37 ° C. and 5% CO ₂ for 4 days. Subsequently, the TEC was infected with avian influenza virus H1N1 at 0.1moi for 24 hours or ConA and PMA were treated at 1,000 × concentration for 16 hours. Then, RNA was extracted from the cells using TRIZOL reagent (Promega, USA). Treated extracted RNA with DNase (Promega, USA) for 1 hour at 37 ℃, then treated with DNaes inhibitor (Promega, USA) to inactivate the DNase, random hexamer (Promega, USA) and M-MLV reverse transcription CDNA was synthesized by performing reverse transcription using an enzyme (SUPERBIO, Korea). PCR was performed using the synthesized cDNA 2ul as a template and using each of the designed primers.

Example  1-3: Cloning  And sequencing

PCR products were electrophoresed on agarose gel to extract a band corresponding to the predicted size (chIFNAR1F1: 662bp, chIFNAR1F2: 1045bp, chIFNAR2F1: 942bp, chIFNAR2F2: 582bp) using a Corebio gel extraction kit (Figure 3). The extracted DNA was cloned into pGEM T-EASY vector (Promega, USA), and the cloned PCR product was inserted through the selection process. At this time, plasmids were obtained from the individual clones in order to confirm that the inserts of the correct size were inserted in the vector, which were digested with EcoRI and electrophoresed on agarose gel (FIG. 4). In addition, the nucleotide sequences of the fragments digested with EcoRI were confirmed to confirm that fragments 1 and 2 of chIFNAR1 (FIG. 5) and fragments 1 and 2 (FIG. 6) of chIFNAR2 were correctly inserted.

For each chIFNAR1 and 2, it was confirmed that the variation of the sequencing sequence was shown by the repeated PCR, and through this, it was confirmed that it is the genetic background of TEC of the chicken used as a template. The genetic background in 8) is specified.

Example  1-4: Subcloning  And sequencing  Confirm

For subcloning into the animal cell expression vector pcDNA vector, each fragment insert of the obtained pGEM clones was obtained by digestion with restriction enzymes, which were subcloned into the pcDNA3.1a vector. First, for chIFNAR1, pGEM chIFNAR1F1 was cut with BamH1 and Stu I, pGEM chIFNAR1F2 was cut with Stu I and Xho I to obtain respective inserts, and each insert was subcloned into pcDNA 3.1a vector cut with BamH1 and XhoI. It was. In addition, for chIFNAR2, both pGEM chIFNAR2F1 and pGEM chIFNAR2F2 were cut with BamH1 and Sac I to obtain respective fragments, which were formed into one fragment using the T4 ligation kit (TAKARA, Japan), followed by Kpn I and Subcloned the pcDNA vector digested with Xho I.

For each of the clones obtained through the selection process, each of the clones were introduced into each clone, at this time, in order to confirm the inserts introduced in each clone, the plasmid obtained from each clone was cut with restriction enzymes and agarose gel It was confirmed by electrophoresis (Fig. 9). In addition, the base sequence of each of the cleaved inserts was determined to further confirm whether the correct insert was obtained (FIGS. 10A and 10B).

Example  2: expression test

Example  2-1: HeLa  To the cell pcDNA chIFAR1  And 2 introducing each plasmid vector Immunofluorescence Assay  Check through

2 μg of DNA was introduced into HeLa cells 1.5 × 10 ⁵ cells using Viva magic reagent (Vivagen, Korea) for 48 hours. Thereafter, it was fixed at room temperature for 30 minutes using 2% formalin and blocked using 10% goat serum. The primary antibody was treated by diluting 1: 2,000 anti-His Ab in 3% goat serum, and detected by confocal microscopy using goat's IgG FITC-conjugated anti-mouse antibody (Molecular probe, US). His-tagged chIFNAR1 and 2 signals were confirmed by the prepared pcDNA 3.1a vector, thereby verifying that they were expressed in the cytoplasm of HeLa cells (FIG. 11).

Example  2-2: HEK -293T cell  on pcDNA chIFNAR1  And 2 introducing each plasmid vector, Western Blot  Check through analysis

6 μg of DNA was introduced into CaK ₂ and HEPES buffered saline (HBS) in 5.0 × 10 ⁵ cells of HEK-293T cells. After 48 hours, the cells were obtained, and 30 ° C at 4 ° C using RIPA buffer (150 mM NaCl, 1% NP40, 0.5% deoxycholic acid, 50 mM Tris-HCl pH8.0, 0.2 mM PMSF, protease inhibitor cocktail) Dissolved for 1 min, centrifuged for 10 min under conditions of 13,200 rpm and 4 ° C. to obtain supernatant, which was subjected to SDS-PAGE, followed by antibody 1 (anti-His 1: 5,000, anti-β actin 1 : 10,000) and signals of chIFNAR1 and 2 His tagged by pcDNA 3.1a vector by Western blot using antibody No. 2 (HRP-conjugated anti-mouse IgG 1: 5,000) and fluorescence induction with ECL solution It was confirmed that it was properly expressed by confirming (Fig. 12).

Example  3: antiviral efficacy assay

Example  3-1: pcDNA chIFNAR1  And 2 each DNA  In chick cells using plasmids IFNAR  Gene Overexpress  Induction test for nonspecific resistance to avian influenza virus

The 10-day-old fertilized eggs were received from the National Livestock Science Institute, TEC was isolated, incubated under conditions of 37 ° C. and 5% CO ₂ for 4 days, and then 2 μg of DNA was introduced using Viva magic reagent. After 48 hours, chicken IFN-α / β were treated for 24 hours each to further amplify the activity of IFNAR. In this case, as the IFN-α / β of the chicken, the culture supernatant obtained after introducing the DNA of each pcDNA chIFN-α / β into HEK-293T cells for 48 hours. After treatment with IFN-α / β, avian influenza virus H9N2 was infected with 0.1moi for 24 hours, western blot was performed using infected cell lysate, and viral protein signal was obtained using antiserum of chickens infected with H9N2. It was confirmed. As a result, it was confirmed that overexpression of IFNAR1 and 2 inhibited the growth of the virus and amplified the antiviral effect by chIFN (FIG. 13A).

In addition, Western blot analysis was applied to a computer program (quantity one software, Bio-Rad Laboratories, USA) to quantitatively analyze the density of each band, and overexpression of IFNAR1 and 2 alone inhibited virus propagation by nearly 35%. And, when simultaneously treated with IFN it was confirmed that the inhibitory effect is further increased by 10 to 20% (Fig. 13B).

Example  3-2: In the cells of chicken chIFNAR1  And 2 Overexpress  Recombination to quantitatively analyze the degree of inhibition of virus proliferation NDV - GFP  Analyzes inhibition pattern using virus

The 10-day-old fertilized eggs were received from the National Livestock Science Institute, TEC was isolated, incubated under conditions of 37 ° C. and 5% CO ₂ for 4 days, and then 2 μg of DNA was introduced using Viva magic reagent. After 48 hours, chicken IFN-α / β were treated for 24 hours each to further amplify the activity of IFNAR. After 24 hours of treatment with IFN-α / β, recombinant NDV-GFP virus expressing GFP at infection was introduced with TCID50 0.1 for 24 hours. Thereafter, the cells were trypsinized and flow cytometry analysis was performed to measure the proportion of GFP-positive cells in the total cells, thereby confirming the inhibition pattern against viral infection (FIG. 14). As a result, it was confirmed that overexpression of IFNAR1 and 2 alone showed 40% inhibition rate and 20-30% additional inhibition effect when simultaneously treating chIFN-α and β.

<110> SUNGKYUNKWAN UNIVERSITY Foundation for Corporate Collaboration <120> A Composition for Preventing Bird Flu Comprising Interferon          Receptor gene <130> PA110114 / KR <160> 10 <170> Kopatentin 1.71 <210> 1 <211> 1707 <212> DNA <213> IFNAR1 <400> 1 atgtatgctt gtgcttctgg gcggctagcg gctgtgctgc tttgtgtctt ggtcgtggtg 60 tcccggtgct gtgcaggcca gactaatctg aagagtcctc aggatattca ggtttatgct 120 gtaaatacaa atttcaccct aatgtggaac tacactggag atggtaccaa cgtgacattt 180 tcagcacaat accagtgctt tgatgatctt cagacaagtg aaccagaatg gaaggaatta 240 tctggttgcc agaatgtcag tcacacagaa tgtgactttt cttcagcaat aactgcatac 300 tatgatacgc atcacatccg cataagggct gaaagaaggg aagccaagtc tccatggtct 360 agcattttcg aaatgattcc gtatgaaata gctcagattg gtccccctga aatagcgttg 420 cagtccataa atggagccat taaaattaac atttctcctc cagaagcaaa tcaggttcga 480 aaaatgtggc taatttctgt gttttttaaa tacaatgtag ttatctggga caattcatcc 540 aatgtagaaa aggtcagaag tattttaccc attgatgtaa taaatgatct tgctccagag 600 actacctatt gtttgaaagt tcaagcaact gttcctctgg aggataaagg aggcctgttc 660 agtccaattc attgcataaa aaccacccgt aaagtaaatg accttctttg cccaacaaat 720 gtgagagttt ttgctttgaa catgaaattc tatctgcttt gggataatca ctataacgaa 780 catgtgacct ataccgtaca gtatctcact gggtacctaa agaaccttta tgatgattac 840 tcctcaaagt ggcagaaggt atcaggatgt gagaacatca ctagcatgaa atgcaatttg 900 tcatctgtca tcaaacatac ttcggcatct tattacttcc gtgtgcaggc tatgaatgaa 960 tataataaat catgtttgtc taaagacgta gaagtagatc ctccggtaac aaatgaaatt 1020 ggccctcctg atgtaaaggt ggacatcagt gatgttttgc tccatatcaa gattactcct 1080 ccaggaggac ctgggaataa aatcatgagt gacctttatg atttctctta ccagattctg 1140 tattggaaga attcatcaga taatgaggag gaagtaaaaa tgaaagaaac aaaacagaca 1200 atagcgacag tctctgatct ggcaccctcg actttgtact gtgtgaaagt acaagcgttc 1260 tcagaagctt acaacaaaag cagtgatttc agcagagagg aatgcatcgg aacagctggc 1320 ggtaaacact tacctctgat cattttagca acatttgcag gtgcgctgac tgttgtcttg 1380 attgtggcat cactggtcat ttttttcctg tatcaagtct ataataaaat caagtacatg 1440 ttctttcctt catgccagac tcctctgaac atagagggct tcggagcaca gctctttagc 1500 agtccatttg tgcctactgt agaagaaccg gtagaaattt gttacataat tgagagtagg 1560 atcacagaag aagtaaatca aattgacttt aaagacaaca aacattttaa acagagcagt 1620 cgagattcag ggaattattc ttacgacgat aatacctcac aaagcaaagg gtcagaagaa 1680 acactaggaa atgacatact actcgag 1707 <210> 2 <211> 1530 <212> DNA <213> IFNAR2 <400> 2 atggaatcac tgatgattgg cccacttcgg tttactcagc ttgtgttcgt cagcattctg 60 tgtgcggctt gttacagcag cttgcctgaa aagattccga gagagccacc tgataatcta 120 caaatgacat ctaacaactt tcagcacatt ttgtcctggc gggcacacaca tgatccaact 180 gtgccaacat actatcgtgt actgtacagt agccacagta actggaagat tgcgaaacag 240 tgttcacgaa ttgtacagcc cttctgtaac ctgacagatg attttcaagt tgtgtctgat 300 gaatattctg cattcgttca aagctttgta ggaactgaag tattcaattc ctctttactc 360 catttctcac cgctctccga gacattcttg ggacctccag aatttaatct cagttcctgt 420 gtccactgca taaatatcac cataaagctg ccacccactc acttaagaaa aaatggaaag 480 ctgctgtctt tatttgatat atataacaaa gttaattatg aaataacact gagaacagtt 540 ggtgaagaac ataagaggtc acctgagaaa gtcactgaag aaccttttag tatagtcatt 600 gaagaattgt atccgaatag aaattactgt gtttctgtta tggtcactgc atctctaaat 660 aaacattcca tcccatcagc ctggaaatgc ataactacag actctgtagc tgagaaagat 720 tattatggca tcacaattgc aggtgctata tgtttttcaa ttattttagt tgtcatcctg 780 aaatgtttgc atttaggagg ttatattctt cacaagaaat cacttccaga tactttgata 840 ttcacgaaga tgtttagcta tttaccgttt acatttgaat gtgaagaaat aacctctgta 900 gagatcattt ataaagaggt taaaaaaaag gcagaaggat ccgttggagc tgtcagcagt 960 gaagatgaca gcgatgacag tgaaagcgat gccatgagta atcatgacta cacaaggcgt 1020 gatatcgtac gtagagcacc tcagtcttct gacacgtctc ctgtatttgt gcagcactct 1080 acgagtagta catgtgatga cagtagcagc tgggtgagtc aaaatccaga tgatggccca 1140 gaagtctttg aagaaaacga gatggatgcc gaagaagaga aagacacgga tagtgagtta 1200 ctcagtcctt tgtccaaggt aaattgtacc tattctctta ggtcaaggag caatttttgc 1260 tttaccataa acttaaaatc tgtgcttctg ggaatctctg aagagaccac agatagttct 1320 gcagctctcc tctcttccca agaagatgct gttgactggc aatgtgcaca tgctgttgaa 1380 tcaaaactcc ttgatgacac agaaagcatg cagaaaccac atcatctaaa caactctgat 1440 gtgtggcaga attcatctgg ttcttccagt gaaagcgact catcggattc agatatggac 1500 ccaaaaagtg aatacgtacg aagactcgag 1530 <210> 3 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 ggatccgccg ccaccatgta tgcttgtgct tctgggc 37 <210> 4 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 gaacaggcct cctttatcc 19 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 ggataaagga ggcctgttc 19 <210> 6 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 ctcgagtagt atgtcatttc ctagtgtt 28 <210> 7 <211> 55 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 ggtaccgccg ccaccatgga atcactgatg attggcccac ttcggtttac tcagc 55 <210> 8 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 ccaacggatc cttctgcc 18 <210> 9 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 ggcagaagga tccgttgg 18 <210> 10 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 ctcgagtctt cgtacgtatt cactttt 27

Claims (10)

A pharmaceutical composition for preventing avian influenza, comprising a polynucleotide encoding a receptor of an interferon or a vector containing the polynucleotide as an active ingredient and a pharmaceutically acceptable carrier.
According to claim 1, wherein the polynucleotide encoding the receptor of the interferon is an interferon (α, β, ο) receptor 1 (IFNAR1) gene or interferon (α, β, ο) which is interferon (α, β, ο) receptor 1 IFNAR2 (interferon (α, β, ο) receptor 2) gene that is receptor 2.
The composition of claim 2, wherein the IFNAR1 gene is represented by the nucleotide sequence of SEQ ID NO: 1.
The composition of claim 2, wherein the IFNAR2 gene is represented by the nucleotide sequence of SEQ ID NO: 2.
A polynucleotide encoding the receptor of the interferon or a vector containing the polynucleotide as an active ingredient, and a bird flu prevention DNA vaccine comprising a pharmaceutically acceptable carrier.
The method of claim 5, wherein the polynucleotide encoding the receptor of the interferon is an interferon (α, β, ο) receptor 1 (IFNAR1) gene or interferon (α, β, ο) is interferon (α, β, ο) receptor 1 The vaccine of IFNAR2 (interferon (α, β, ο) receptor 2) gene that is receptor 2.
The vaccine of claim 6, wherein the IFNAR1 gene is represented by the nucleotide sequence of SEQ ID NO: 1.
The vaccine of claim 6, wherein the IFNAR2 gene is represented by the nucleotide sequence of SEQ ID NO: 2.
A polynucleotide represented by the nucleotide sequence of SEQ ID NO: 1 or 2, a vector comprising the polynucleotide, a composition comprising the polynucleotide or the vector, or a DNA vaccine comprising the polynucleotide or the vector is administered to an individual except a human. Comprising a step of preventing avian influenza.
The method of claim 9, wherein the subject is poultry.

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