KR101418130B1 - Fluorescence tagged influenza virus and method for producing thereof - Google Patents

Abstract

The present invention relates to a fluorescence-tracing recombinant influenza virus comprising a base sequence containing a cysteine residue in a part of an influenza virus gene. In detail, the present invention relates to a method for producing the recombinant influenza virus, a method for treating a cell transfected with the recombinant influenza virus with FLASH-EDT ₂ , which is a fluorescent substance, The present invention relates to a method for analyzing the action mechanism of an antiviral agent by treating a transfected cell with a fluorescent substance FLASH-EDT ₂ and treating the antiviral agent.

Flu virus, fluorescent material, reverse genetics engineering method, cell image

Description

FIELD OF THE INVENTION [0001] The present invention relates to a fluorescence-

The present invention relates to a fluorescence-tracing recombinant influenza virus comprising a base sequence containing a cysteine residue in a part of an influenza virus gene. More particularly, the present invention relates to a method for producing the recombinant influenza virus, a method for treating a cell transfected with the recombinant influenza virus with FLAsH-EDT ₂ as a fluorescent substance to track fluorescence in the cell, The present invention relates to a method for treating an antiviral agent by treating FLASH-EDT ₂ , a fluorescent substance, with an antiviral agent.

Influenza viruses are not only people but also respiratory infectious disease viruses widely spread in domestic animals such as birds, pigs, horses, seals, whales, minks and many other animals. In some cases, they are infectious viruses that can cause enormous damage. Influenza viruses are mainly infectious in the respiratory system, but they can penetrate into the lungs and cause fatal diseases. Influenza caused by influenza virus is an acute respiratory disease accompanied by upper and lower respiratory symptoms, fever, headache, muscle aches and helplessness.

Influenza virus is a virus belonging to the genus Orthomyxoviridae and having eight negative sense RNA fragments of PB2, PB1, PA, HA, NP, NA, M and NS. Among them, two proteins hemagglutinin (HA) and neuraminidase (NA), which constitute the envelope protein, are important immunogens for inducing immunological antibodies, The influenza viruses are classified into various subtypes according to the types of HA and NA. The number of subtypes revealed to date is 16 types of HA and 9 types of NA .

Influenza usually presents with various symptoms and severity according to the subtypes that are prevalent in winter. It is highly infectious and can cause complications of pneumonia and cardiopulmonary disease, resulting in high mortality. Therefore, not only the elderly, children, Vaccination and antiviral medication are strongly recommended.

In particular, the pandemic flu virus, which occurs worldwide every 10 to 40 years, is very lethal, like the Spanish flu virus that killed more than 20 million people in 1918. After the Hong Kong flu virus of the late '60s, , It is expected that a fatal Pandemic flu virus will appear in the near future. One of the candidates for pandemic influenza, avian influenza virus, is an acute viral disease that is infected with chicken, duck, turkey and wild birds. There are a variety of viruses ranging from no pathogenicity to 100% highly pathogenic, and the highly pathogenic avian influenza virus It is a Class 1 infectious disease and is classified as List A in the OIE (International Bureau of Waters). The avian influenza virus is a common infectious virus that spreads by droplets, air and water, but the main cause of the spread is contaminated feces. That is, feces can spread on human hands, feet, feed carts, utensils, and equipment. In addition, the avian influenza virus may infect humans through intermediary hosts such as pigs and chickens. When the avian influenza virus causes gene mutation or recombination with other viruses in the intermediate host, It spreads to people through infection and causes flu. In addition, in the case of the H1N1 strain of influenza, which is currently prevalent in the world, the mortality rate is significantly lower than that of avian influenza but it is highly contagious. The development of effective preventive vaccines against these newly emerging influenza viruses is inadequate and the limitations of current therapeutic agents remain a problem.

Four drugs, including Amantadine, Rimantadine, Oseltamivir and Zanamivir, have been approved by the US Food and Drug Administration for the treatment of influenza viruses. received. Amantadine and rimantadine have been used for over 40 years and have been reported to be easily tolerated. In the case of oseltamivir and zanamivir, relatively recently developed and relatively effective, both have the same mechanism of inhibiting the enzyme activity of NA, the outer protein of influenza virus, (Mai Le, Q., et al., Nature 437: 1108, 2005). However, there is a high possibility that a strain resistant to several mutations may occur, The development of anti-flu virus drugs is urgent.

To date, many researchers have tried to identify the lifecycle of intracellular viruses and to find out the interaction between viruses and cells. One of the ways to identify interactions between these viruses and cells is single-virus tracking within the cell. This single virus tracing allows the dynamic interaction of each viral particle and cell structure and these interactions to reveal the infectious stage of the virus that we did not know. This single virus tracing has revealed mechanisms of intracellular entry, trafficking, and excretion of several animal viruses (Brandenburg, et al., Nature 5: 197-208, 2007) Can be found. Transport of organelles or macromolecules called cargo is mediated by cytoplasmic microtubules. Defects in such cellular transport cause serious disease. This cytoplasmic microtubule allows the virus to reach a specific location for gene replication and also helps release the newly generated virus (Greber, et al., Cell 124; 741-754, 2006). Based on this concept, in 2003, Harvard University used influenza virus X-31 as a lipophilic fluorescent dye, 1,1'-dioctadecyl-3,3,3 ', 3'-tetramethylindodicarbocyanine (DiD) (Lakadamyali, et al., PNAS 5; 9280-9285, 2003). In addition, it was surprisingly found that the cytoplasmic part of the cell membrane But the fusion of the virus around the nucleus was found.

Through the single virus tracking as described above, it is possible to know the interaction between viruses and cells that were not known before, and new antiviral mechanisms can be revealed from such knowledge. In addition, it is also important to attach a fluorescent substance to a single virus tracking to create a virus for tracking fluorescence. It is difficult to introduce large-scale proteins such as green fluorescence proteins into viruses when producing viruses for fluorescent tracing, and it is preferable to use those that are small in size and do not affect virus growth when inserted into virus . In this respect, since it is desirable to attach a fluorescent dye to a virus and to perform fluorescence tracking, there have been many attempts to trace a single virus using fluorescent dyes. One of them is the virus tracking using DiD described above, and the adenovirus is also tracked using FITC or Texas red (Pombo, et al., EMBO 13 (21); 5075-5085 1994) Cy5 was used to observe the infection of adeno-associated virus in real time (Seisenberger, et al., Science 294; 1929-1932 2001).

Accordingly, the present inventors have made intensive efforts to track a single virus using a fluorescent dye. As a result, it has been found that FlAsH-EDT ₂ does not fluoresce through binding with lipids in the membrane portion of a virus like other fluorescent dyes (DiD) And covalently binds to specific proteins of viruses. Therefore, it is possible to identify specific proteins of viruses, and it is ideal for single virus tracking because of its good membrane permeability. Thus, it has been found that the cysteine residue And a new fluorescence-tracing influenza virus was prepared by a reverse genetics method to confirm the virus in the cell by binding the fluorescent substance, thereby completing the present invention.

It is an object of the present invention to provide a fluorescence-tracing recombinant influenza virus comprising a base sequence containing a cysteine residue in a part of an influenza virus gene.

It is still another object of the present invention to provide a method for the production of a virus, comprising: (a) introducing a cysteine residue into a viral gene selected from the group consisting of HA (hemagglutinin), PB2, PB1, PA, NP, NA, And (b) transfecting the gene into which the cysteine residue has been introduced, together with genes not selected in step (a), into a host cell to produce a virus. .

It is yet another object of the present invention to provide a method for fluorescence tracing an intracellular flu virus, comprising the step of treating a transfected cell with a fluorescent substance FLASH-EDT ₂ .

It is still another object of the present invention to provide a method for producing a recombinant influenza virus, which comprises treating a transfected cell with FLASH-EDT ₂ , which is a fluorescent substance, And simultaneously or sequentially treating the cell with an antiviral agent. The present invention also provides a method for analyzing the action mechanism of an antiviral agent.

In order to achieve the above object, in one aspect, the present invention relates to a fluorescence-tracing recombinant influenza virus comprising a base sequence containing a cysteine residue in a part of an influenza virus gene.

The term "influenza virus " in the present invention refers to a virus belonging to the genus Orthomyxoviridae and having eight negative sense RNA fragments of PB2, PB1, PA, HA, NP, it means. Among them, two proteins hemagglutinin (HA) and neuraminidase (NA), which constitute the envelope protein, are important immunogens for inducing an immune antibody, Influenza viruses are classified into various subtypes according to the types of HA and NA. There are 16 types of HA and 9 types of NA.

The influenza virus is not limited to any viruses having eight negative sense RNA fragments of PB2, PB1, PA, HA, NP, NA, M and NS such as H3N2, H5N1 and H1N1, to be.

The term "cystein " in the present invention means a sulfur-containing amino acid present naturally in small amounts in many proteins, and cysteine contains only thiol group (-SH) among 20 basic amino acids. In the present invention, an external cysteine residue is inserted into a part of an influenza virus gene so as to bind with FLASH-EDT ₂ , a fluorescent substance known as a substance that binds to a cysteine residue (CCRECC) and emits fluorescence. The fluorescent material FLASH-EDT ₂ The Unlike other fluorescent materials, it does not fluoresce through binding with lipids in the membrane of virus, but fluoresces through covalent bonds with cysteine residues, so it is possible to identify specific proteins of virus.

In a preferred embodiment, the cysteine residues can be two or more, preferably two to six, more preferably four, and most preferably tetracysteine motifs (CCXXCC, Cys4).

The term "for fluorescence tracing" in the present invention is a method for detecting the interaction between a virus and a cell, which is designed as a method for tracking a single virus in a cell. The cysteine residue capable of binding to a fluorescent substance , Which means that fluorescence can be displayed when a fluorescent substance is treated to track the movement of a virus protein such as observing tracking of a virus protein.

The fluorescent substance for fluorescence tracking of the present invention is a dye capable of binding with a cysteine residue and is not limited as long as it is capable of binding fluorescence by binding with a cysteine residue, but is preferably FLAsH-EDT ₂ .

The term "FLASH-EDT ₂ " (4 ', 5'-bis (1,3,2-dithioarsolan-2-yl) fluorescein- (1,2-ethanedithiol) 2)) means a substance which fluoresces through a covalent bond with a cysteine residue , And it is a substance having good permeability.

In one preferred embodiment, the influenza virus gene is selected from the group consisting of HA, PB2, PB1, PA, NP, NA, M and NS, preferably HA gene, more preferably C - terminal portion, most preferably the nucleotide sequence of SEQ ID NO: 5.

In one preferred embodiment, the recombinant influenza virus is a recombinant influenza virus with accession number KBPV 2009-001, comprising four cysteine residues, based on the H1N1 influenza virus.

The present inventors confirmed the activity and fluorescence expression of the fluorescent-tracing recombinant influenza virus according to the present invention in the following manner. In a specific example, the HA gene, the surface protein of A / PR / 8/34, the H1N1 type of human type A influenza virus including four cysteine residues in the tag for fluorescent tracing, was amplified using primers of SEQ ID NOS: 1, 2, 3 and 4 , Which was confirmed by DNA sequencing (Example 1). The HA gene fragment containing the cysteine residue as a tag for fluorescent tracing was inserted at the position of BsmB1 of the vector pHW2000, and the vector was named C4-HA (Fig. 1).

The vectors in which the remaining genes of the parent virus PB2, PB1, PA, NP, NA, M, and NS were inserted were transfected together with the C4-HA vector to 293-T cells as host cells, The preparation of the inserted recombinant influenza virus was performed, and the prepared virus was confirmed by plaque analysis (Example 2).

In order to investigate whether the cysteine residue affects the activity of the virus due to the insertion of the cysteine residues, the present inventors infected MDCK cells with the wild type A / PR / 8/34 virus and the recombinant toxin virus prepared as described above, As a result of the analysis, it was confirmed that there was no difference in growth activity between the wild type virus and the recombinant influenza virus. Thus, it was confirmed that there was no influence on the activity of the virus due to the insertion of the cysteine residue. (Fig. 3). ≪ tb > < TABLE >

In addition, in order to confirm that the inserted cysteine residue tag fluoresces well in the cells, the prepared recombinant influenza virus and FLASH-EDT ₂ were mixed at room temperature, the cells were infected and cultured for 72 hours, Fluorescence was observed in the group treated with recombinant virus for fluorescence tracking and FLASH-EDT ₂ (FIG. 4 (A)), and FLAsH-EDT ₂ (FIG. 4 (B)), it was confirmed that the fluorescence was well expressed in the cells, and the fluorescence-tracing recombinant influenza viruses produced in the present invention were found to have intrinsic activity of the virus And fluorescence was expressed without any influence.

(A) introducing a cysteine residue into a viral gene selected from the group consisting of HA (hemagglutinin), PB2, PB1, PA, NP, NA, M and NS; And

(b) a step of transfecting the gene into which the cysteine residue has been introduced, together with genes not selected in step (a), into a host cell to produce a virus. will be.

The method of introducing the cysteine residue of the step (a) into the viral gene can be used without limitation to molecular biology methods known in the art such as chemical synthesis and enzymatic method, but is preferably a method using RT-PCR. A virus gene fragment into which the cysteine residue has been introduced, and the remaining seven parental viral genes are transferred to the host cell. The vector is known in the art and is not limited thereto, preferably pHW2000.

PB1, PA, HA, NP, NA, M, and NS, which are eight genes constituting the influenza virus, can be used without restriction, preferably HA gene, RT-PCR is performed on the HA using the primers of SEQ ID NOS: 1, 2, 3 and 4 to introduce four cysteine residues at the C-terminal position of the HA gene.

The HA gene is a GenBank accession number (EF467821.1), and a motif in which four cysteines of SEQ ID NO: 5 are present at positions 1727 to 1756 of the C-terminal position of SEQ ID NO: 6 was subjected to RT- Respectively.

The transfection of step (b) may be carried out by a method selected from the group consisting of calcium phosphate-DNA coprecipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposomal fusion, lipofectamine and protoplast fusion And the like can be carried out by various methods known in the art.

Host cells are host cells capable of producing viruses by infecting viral genes and include eukaryotic cells such as yeast, fungi, protozoa, higher plants, insects or amphibian cells, or CHO, HeLa, HEK293, 293T, NIH / 3T3 , COS-1, COS-7, CHO-K1, Hep G2, and mammalian cells including humans, which are commonly used in the art. Preferably 293T.

When the FLASH-EDT ₂ is treated in a cell, the produced recombinant control virus for fluorescence tracking does not lose the activity of the virus and expresses the fluorescence. Those skilled in the art will appreciate that the above- Recombinant influenza virus can be easily produced. The present inventor has found that a recombinant virus for tracing fluorescence in which four cysteine residues are introduced into the C-terminal of HA of H1N1 is deposited with the Bank of Korea Virus Bank (Seoul, Korea) No. KBPV-2009-001, and those skilled in the art will be able to readily obtain and reproduce the invention at any time (FIG. 5).

In another aspect, the present invention relates to a method for fluorescence tracking an intracellular flu virus, comprising the step of treating a transfected cell with a fluorescent substance FLASH-EDT ₂ .

In a preferred embodiment, the recombinant influenza virus-transfected cells are selected from the group consisting of: (a) designing a PCR primer that attaches a cysteine residue to the HA gene genome of the influenza virus; (b) performing a PCR reaction using a PCR primer that attaches a cysteine residue to the HA gene cDNA; And (c) transfecting the seven gene plasmids of the HA gene plasmid with cysteine residues and PB2, PB1, PA, NP, NA, M and NS.

In one preferred embodiment, the method of fluorescence tracing the intracellular recombinant influenza virus may relate to a method of fluorescence tracing of intracellular migration of viral proteins.

In one preferred embodiment, the method of fluorescence tracing the intracellular recombinant influenza virus may relate to a method of fluorescence tracing the interaction of virus with cell organelles. The new antiviral mechanism will be known by fluorescence tracking of the interaction with the cell organs in real time as described above.

In a preferred embodiment, the method of tracking the fluorescence of the intracellular recombinant influenza virus may be a method of real-time fluorescence tracking of the interaction between virus proteins and host cells.

In another aspect, the present invention provides a method for producing recombinant influenza virus, comprising the steps of: treating a transfected cell with a recombinant influenza virus FLASH-EDT ₂ as a fluorescent substance; And a method for analyzing the action mechanism of an antiviral agent, comprising simultaneously or sequentially treating an antiviral agent to the cell.

The antiviral agent controls the proliferation or infection of the virus through the action of suppressing or increasing each step such as a certain drug process, a signal transduction system, a post-transcriptional regulatory process, a replication regulatory process, Refers to an agent screened as a therapeutic agent and includes, but is not limited to, all types of antiviral agents known in the art. The antiviral agent can be simultaneously or sequentially treated to determine the suitability of the screened antiviral agent through real-time tracking of the cysteine-tagged recombinant influenza virus, which is a mechanism of action of the antiviral agent.

The influenza viruses were prepared by introducing the cysteine residues for fluorescent tracing into each of the influenza virus genes using the fluorescent-tracing influenza virus designed and manufactured in accordance with the present invention and the retrospective platform for producing the same, The intracellular movement of proteins can be observed in real time. In particular, the fluorescence-tracing influenza virus of the present invention will be able to observe the interaction between intracellular viruses and cell organelles in real time and obtain new knowledge about interaction between each virus protein and host cell And is expected to be usefully used to elucidate the mechanism of action of screened antiviral agents.

Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are only for illustrating the present invention, and the scope of the present invention is not construed as being limited by these embodiments.

Example  1: for fluorescence tracking tag Introduced HA  Gene Cloning

The influenza virus A / PR / 8/34 (H1N1) was injected into a single cell layer of MDCK (ATCC Cat. # CCL-34) (multiplicity of infection), followed by incubation at 37 ° C for 8 hours. After the culture solution was removed, only the cells were harvested and centrifuged at about 10,000 xg for 10 minutes to obtain a cell fraction. Total RNA was isolated using Easy Blue kit of Intron Bio (Seoul, Korea) The absorbance was measured at ㎚ to determine the concentration of extracted RNA.

RT-PCR was performed using 2 쨉 g of extracted total RNA and the primers of SEQ ID NOS: 1, 2, 3 and 4 shown in Table 1 below.

HA-C4 F1 (SEQ ID NO: 1) 5'-AGCCGTCTCTGGGAGCAAAAGCAGGGGAAAATAAAAAC-3 ' HA-C4 R1 (SEQ ID NO: 2) 5'-TCAGCAGCACTCCCTGCAGCAGCCGGCGCCGGCGATGCATATTCTGCACTGCAAAG-3 ' HA-C4 R2 (SEQ ID NO: 3) 5'-GGGTGTTTTTCCTCATATTTCTGAAATTCTAATCTCAGCAGCACTCCCTGCAGC-3 ' HA-C4 R3 (SEQ ID NO: 4) 5'-AGCCGTCTCATATTAGTAGAAACAAGGGTGTTTTTCCTCATATTTC-3 '

Reverse transcription-polymerase reaction and PCR reaction were performed by BIO-RAD's DNA Engine Thermal Cycler was used. The amplification program started with 1 cycle (1st strand cDNA synthesis) for 45 minutes at 37 占 폚 and 1 cycle (inactivation of the reverse transcriptase and cDNA denaturation) at 94 占 for 2 minutes. The PCR reaction cycle was repeated for 30 cycles (PCR amplification) at 94 ° C for 20 seconds, 55 ° C for 30 seconds, and 72 ° C for 1 minute and 45 seconds, followed by extension at 72 ° C for 5 minutes to 1 cycle . The PCR product was analyzed by agarose gel electrophoresis to confirm the length of the amplified DNA. The presence or absence of the introduced cysteine residue using the primer was confirmed by DNA sequencing. The gene HA region of SEQ ID NO: 6 contains the nucleotide sequence of SEQ ID NO: 5 from SEQ ID NOs: 1727 to 1756, which was inserted at the BsmB1 site of the vector pHW2000 (FIG. 1) and the vector was designated C4-HA.

Example  2 : Transformations  Production of virus through

The HA gene, the surface protein of the A / PR / 8/34 virus, H1N1 of the human influenza A virus, was amplified by PCR using a vector containing the cysteine residue and a vector (PB2, PB1, PA Each vector was transfected into 1 × 10 ⁶ 293T cells in a 293-T cell line using Lipofectamine (Invitrogen, Carsbad CA) according to the protocol of the manufacturer. I was shocked. After incubation for 2 days in a 37 ° C CO ₂ incubator, plaque assay was performed to confirm the production of the virus. When the production of the virus was confirmed, the virus was isolated from the plaque, and the virus was inoculated into the urothelium of the chicken embryo cultured for 11 days, and the embryo was further cultured at 37 ° C for 3 days to obtain a sufficient amount of virus . The amount of virus was measured by the hemagglutinin agglutination (HA) assay, and the virus was cultured in the embryo. The virus was divided into 1.5 ml tubes and stored in a refrigerator at -80 ° C. until the next experiment.

Example  3: Measurement of activity of flu virus

In order to measure the activity of the produced fluorescence-tracing influenza virus, the wild-type A / PR / 8/34 virus and the virus prepared in the present invention were cultured in chicken embryos and subjected to the erythrocyte agglutination reaction and plaque analysis, . Red blood cell agglutination reaction was performed by diluting virus twice in 96-well plate with 1% chicken red blood cells and reacting at 4 ° C for 30 minutes. Plaque assays were performed by culturing MDCK cells in 6-well plates (containing 1x10 ⁶ cells per well) Viruses serially diluted 10-fold were infected at room temperature for 45 minutes. The virus solution used for the infection was removed and the cells were washed with 1 ml of PBS (Phosphate Buffered Saline) and then washed with an overlay medium (1% agarose, DMEM containing trypsin enzyme at a concentration of 10 μg / Dulbecco's Modified Eagle's Medium, GIBCO Invitrogen Coporation, USA). After the overlay medium had hardened, it was cultured in a 5% CO ₂ incubator at 37 ° C for 72 hours.

As shown in FIG. 3, activity of the virus was not significantly different from that of the virus A / PR / 8/34, which is the parental virus, by the hemagglutination reaction method and plaque analysis. Suggesting that the cysteine residues in the HA portion of the surface protein do not significantly affect the activity of the virus.

Example  4: Measurement of Fluorescence-Induced Flu Virus Activity in Cells

To observe the fluorescence expression of the fluorescence tracking virus, 10 ³ PFU of virus and 1.25 μM of FlAsH-EDT ₂ (Invitrogen, Carsbad CA) were mixed at room temperature in the state of blocking light, reacted for 1 hour, and then infected in a 6-well plate at room temperature for 45 minutes. After the virus solution used for infection was removed, the cells were washed with 1 ml of PBS, DMEM medium containing trypsin enzyme at a concentration of 5 μg / ml was added and incubated for 72 hours in a 5% CO ₂ incubator at 37 ° C. During the incubation, the fluorescence of the virus was confirmed by fluorescence microscopy.

As a result, it was confirmed that fluorescence was observed in the cells with the fluorescence-tracing influenza virus introduced with the FlAsH-EDT ₂ and the fluorescence was not observed in the FlAsH-EDT ₂ cells treated with the control group 4).

FIG. 1 is an illustration of a cloning method for the fluorescence-tracing flu virus FIIV (FlAsH Interacting Influenza Virus). The influenza virus HA gene inserted into the pHW2000 vector through PCR is inserted. The cysteine residue gene is from 1727 to 1756.

2 is a diagram illustrating a virus production method by a reverse genetics method. Eight gene plasmids (including genes with cysteine residues) can be transfected into 293-T cells to generate viruses.

Figure 3 compares the titer of the wild-type A / PR / 8/34 virus with the fluorescence tracking virus. Compared with the wild-type virus, the fluorescence-tracing virus showed no change in titer, confirming that the cysteine residues did not affect viral replication.

Figure 4 relates to intracellular fluorescence expression of the virus for fluorescence tracing. Fluorescence expression can be confirmed by treating FIAsH, a fluorescent dye. (A) is a group treated with recombinant influenza virus and FLASH-EDT ₂ , and (B) is a control group treated with FLASH-EDT ₂ alone.

Fig. 5 is a deposit made with the National Institute of Advanced Industrial Science and Technology, which is the deposit number KBPV-2009-001.

<110> Industry-Academic Cooperation Foundation, Yonsei University <120> FLUORESCENCE TAGGED INFLUENZA VIRUS AND METHOD FOR PRODUCING          THEREOF <130> PA090257 <160> 6 <170> Kopatentin 1.71 <210> 1 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> Primer for HA-C4 F1 <400> 1 agccgtctct gggagcaaaa gcaggggaaa ataaaaac 38 <210> 2 <211> 56 <212> DNA <213> Artificial Sequence <220> <223> Primer for HA-C4 R1 <400> 2 tcagcagcac tccctgcagc agccggcgcc ggcgatgcat attctgcact gcaaag 56 <210> 3 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> Primer for HA-C4 R2 <400> 3 gggtgttttt cctcatattt ctgaaattct aatctcagca gcactccctg cagc 54 <210> 4 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> Primer for HA-C4 R3 <400> 4 agccgtctca tattagtaga aacaagggtg tttttcctca tatttc 46 <210> 5 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> TETRACYSTEINE MOTIF <400> 5 gccggcgccg gctgctgcag ggagtgctgc 30 <210> 6 <211> 1778 <212> DNA <213> HA gene of influenza virus <400> 6 agcaaaagca ggggaaaata aaaacaacca aaatgaaggc aaacctactg gtcctgttat 60 gtgcacttgc agctgcagat gcagacacaa tatgtatagg ctaccatgcg aacaattcaa 120 ccgacactgt tgacacagtg ctcgagaaga atgtgacagt gacacactct gttaacctgc 180 tcgaagacag ccacaacgga aaactatgta gattaaaagg aatagcccca ctacaattgg 240 ggaaatgtaa catcgccgga tggctcttgg gaaacccaga atgcgaccca ctgcttccag 300 tgagatcatg gtcctacatt gtagaaacac caaactctga gaatggaata tgttatccag 360 gagatttcat cgactatgag gagctgaggg agcaattgag ctcagtgtca tcattcgaaa 420 gattcgaaat atttcccaaa gaaagctcat ggcccaacca caacacaacc aaaggagtaa 480 cggcagcatg ctcccatgcg gggaaaagca gtttttacag aaatttgcta tggctgacgg 540 agaaggaggg ctcataccca aagctgaaaa attcttatgt gaacaagaaa gggaaagaag 600 tccttgtact gtggggtatt catcacccgt ctaacagtaa ggatcaacag aatatctatc 660 agaatgaaaa tgcttatgtc tctgtagtga cttcaaatta taacaggaga tttaccccgg 720 aaatagcaga aagacccaaa gtaagagatc aagctgggag gatgaactat tactggacct 780 tgctaaaacc cggagacaca ataatatttg aggcaaatgg aaatctaata gcaccaaggt 840 atgctttcgc actgagtaga ggctttgggt ccggcatcat cacctcaaac gcatcaatgc 900 atgagtgtaa cacgaagtgt caaacacccc tgggagctat aaacagcagt ctccctttcc 960 agaatataca cccagtcaca ataggagagt gcccaaaata cgtcaggagt gccaaattga 1020 ggatggttac aggactaagg aacattccgt ccattcaatc cagaggtcta tttggagcca 1080 ttgccggttt tattgaaggg ggatggactg gaatgataga tggatggtac ggttatcatc 1140 atcagaatga acagggatca ggctatgcag cggatcaaaa aagcacacaa aatgccatta 1200 acgggattac aaacaaggtg aactctgtta tcgagaaaat gaacattcaa ttcacagctg 1260 tgggtaaaga attcaacaaa ttagaaaaaa ggatggaaaa tttaaataaa aaagttgatg 1320 atggatttct ggacatttgg acatataatg cagaattgtt agttctactg gaaaatgaaa 1380 ggactctgga tttccatgac tcaaatgtga agaatctgta tgagaaagta aaaagccaat 1440 taaagaataa tgccaaagaa atcggaaatg gatgttttga gttctaccac aagtgtgaca 1500 atgaatgcat ggaaagtgta agaaatggga cttatgatta tcccaaatat tcagaagagt 1560 caaagttgaa cagggaaaag gtagatggag tgaaattgga atcaatgggg atctatcaga 1620 ttctggcgat ctactcaact gtcgccagtt cactggtgct tttggtctcc ctgggggcaa 1680 tcagtttctg gatgtgttct aatggatctt tgcagtgcag aatatgcatc tgagattaga 1740 atttcagaaa tatgaggaaa aacacccttg tttctact 1778

Claims (14)

Fluorescence-inducing recombinant influenza virus having introduced the nucleotide sequence of tetracysteine motif C-C-R-E-C-C at the C-terminus of the influenza virus hemagglutinin (HA) gene. delete The use according to claim 1, wherein said influenza virus is A / PR / 8/34 (H1N1) virus. The fluorescent substance according to claim 1, wherein the fluorescent substance for fluorescence tracking is selected from the group consisting of 4 ', 5'-bis (1,3,2-dithioarsoran-2-yl) fluorine- (1,2-ethanedithiol) (FLASH-EDT ₂ ), a fluorescent-tracing recombinant influenza virus. delete The fluorescent-tracing recombinant influenza virus according to claim 1, wherein the nucleotide sequence of the motif is SEQ ID NO: (a) introducing a nucleotide sequence of tetracysteine motif C-C-R-E-C-C into a hemagglutinin (HA) gene; And (b) the HA gene into which the nucleotide sequence of the tetracysteine motif CCRECC has been introduced is transformed into polymerase basic 2 (PB2), polymerase basic 1 (PB1), polymerase subunit (PA), nucleoprotein The present invention provides a method for producing a virus by transfecting a host cell with a neuraminidase (NP), neuraminidase (NA), a matrix protein (M), and a nonstructural protein (NS) And the method for producing a recombinant influenza virus for tracing fluorescence according to any one of claims 3 to 4 and 6. 8. The method of claim 7, wherein introducing tetracysteine motif C-C-R-E-C-C into hemagglutinin (HA) comprises performing RT-PCR using primers of SEQ ID NOS: 1, 2, 3 and 4. The recombinant influenza virus according to claim 1, wherein the recombinant influenza virus is selected from the group consisting of 4 ', 5'-bis (1,3,2-dithioarsoran-2-yl) fluorine- (1,2- ethanedithiol) 2) &lt; / RTI &gt; (FLAsH-EDT ₂ ). 10. The method of claim 9, wherein the recombinant influenza virus-transfected cells are selected from the group consisting of: (a) designing a PCR primer that attaches a tetracysteine motif to the hemagglutinin gene genome of influenza virus; (b) performing a PCR reaction using a PCR primer that attaches a tetracysteine motif to a hemagglutinin gene cDNA; And (c) a hemagglutinin gene plasmid with tetracysteine motif, polymerase basic 2 (PB2), polymerase basic 1 (PB1), polymerase subunit (PA), nucleoprotein (NP) ), A neuraminidase (NA), a matrix protein (M), and a nonstructural protein (NS), were transfected together. How to Track Fluorescence. 10. The method of claim 9, wherein the intracellular movement of the viral proteins is fluorescently traced. 10. The method of claim 9, wherein the interaction of the virus with the cell organs is fluorescence tracking. 10. The method of claim 9, wherein the interaction of the viral proteins with the host cell is fluorescence tracking. The recombinant influenza virus according to claim 1, wherein the recombinant influenza virus is selected from the group consisting of 4 ', 5'-bis (1,3,2-dithioarsoran-2-yl) fluorine- (1,2- ethanedithiol) 2) (FLAsH-EDT ₂ ); And simultaneously or sequentially treating the cell with an antiviral agent.

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