KR101997329B1 - Method for Promoting Norovirus Replication - Google Patents

Abstract

The present invention provides a method for increasing replication of human norovirus. The present invention enables the study of infection and disease of norovirus by culturing and propagating norovirus in the laboratory.

Description

Method for Promoting Norovirus Replication

The present invention relates to a method of increasing replication of norovirus.

Norovirus and rotavirus are the main pathogens of viral diarrheal diseases. Noroviruses are the leading cause of enteritis in all ages, sporadically or in groups around the world. In recent years, the infection of norovirus has increased more and more and more, and the importance of it is emerging. According to a recent study of domestic norovirus infection, norovirus infection is most frequent in winter, and 20 genotypes have been identified. It is reported that norovirus GII 4 has the highest infection rate and that norovirus of GII group is higher than that of GI group.

As such, the occurrence of diseases caused by noroviruses is of public health concern, but since noroviruses are not easily cultured in the laboratory, studies on the relationship between contaminants and the resulting norovirus infections and diseases are insufficient.

Therefore, in order to study the relationship between norovirus infection and disease outbreak, in vitro culture methods should be established.

Throughout this specification, many papers and patent documents are referenced and their citations are indicated. The disclosures of cited papers and patent documents are incorporated herein by reference in their entirety, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.

 Se-Ah O et al., Molecular Characterization of Norovirus and Rotavirus in Outbreak of Acute Gastroenteritis in Seoul. Journal of Bacteriology and Virology. 43 (4): 307-316. 2013  Yun-Joo K et al., Characteristics of Norovirus Occurrence in Jeju. Journal of Environmental Science International. 23 (2): 219-229. 2014  Duizer E et al., Laboratory efforts to cultivate noroviruses. J Gen Virol. 85 (Pt 1): 79-87. 2004  Karin Bok et al., Chimpanzees as an animal model for Human norovirus infection and vaccine development. PNAS. 108 (1): 325-330. 2011  Danyelle N., Identification of transferrin receptor 1 as a hepatitis C virus entry factor. PNAS. 110 (26): 10777-10782. 2013  Olivier T., Nucleolin interacts with influenza A nucleoprotein and contributes to viral ribonucleoprotein complexes nuclear trafficking and efficient influenza viral replication. Nature Medicine. 17: 1132-1135. 2011  Erwin Duizer et al. Laboratory efforts to cultivate noroviruses Journal of General Virology 85: 79-87. 2004

We sought to establish a method of increasing replication of norovirus in a laboratory. The present inventors have completed the present invention by confirming that a minute current flows in the human body, and thus a minute current flows in the cells infected with the norovirus to increase the replication of the norovirus.

Accordingly, it is an object of the present invention to provide a method for increasing replication of norovirus.

Another object of the present invention is to provide a method for detecting norovirus.

Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.

According to one aspect of the invention, the invention provides a method of increasing replication of norovirus comprising applying electrical stimulation to a cell infected with norovirus.

The norovirus is a type of virus that causes non-bacterial acute gastroenteritis, and is a family caliciviridae and genus Norovirus virus of Group IV. The noroviruses are classified into GI, GII, GIII, GIV and GV types according to genotypes, among which human noroviruses are GI, GII and GIV.

Specifically, the norovirus is a norovirus whose genotype is GI, GII or GIV. More specifically, the norovirus is a norovirus whose genotype is GII. More specifically, the norovirus is a norovirus of genotype GII-4.

In order to carry out the method of increasing the replication of the norovirus of the present invention, after first culturing the cells, the norovirus must be infected with the cells.

Such cells include various cells with norovirus sensitivity known in the art.

Specifically, the cells are Madein Darby canine kidney (MDCK), Madein Darby bovine kidney (MDBK), Caco-2, Ht-29, HuTu-80, Detroit 551, Detroit 562, HTC-8, MA104 (Microbiological Associates 104 ), AGS, I-407, Vero, HEC, Cotton rat primary epithelial cell (CR-PEC), FRhK-4, HeLa, Kato-3, Crandell Reese Feline Kidney (CRFK) and IEC-6 One or more cells. More specifically, the cell is MDCK.

After culturing the cells under appropriate culture conditions (temperature and atmospheric conditions) for each cell, the norovirus is inoculated into the cells.

Culture temperature conditions of the cells may be 20-40 ℃, more specifically may be 25-40 ℃, 30-40 ℃, 35-40 ℃, and most specifically may be 37 ℃ but is not limited thereto. no.

The conditions for culturing the cells may be 0-10%, 1-9%, 2-8%, 3-7% CO ₂ , and most specifically, 5% CO ₂ , but are not limited thereto. It can be cultured in any CO ₂ condition that can be used conventionally in the art.

The culture of the cells may use a variety of media known in the art suitable for the culture of each cell. The medium is, for example, Fitton-Jackson Modification (BGJb), BME, Brinster's BMOC-3, CMRL, CO ₂ -Independent Medium, DMEM Media, DMEM / F-12 Media, MCDB 131, Media 199, Minimum Essential Media ( MEM), Modified Eagle Medium (MEM), Opti-MEM® I, RPMI Medium 1640, and the like.

In addition, the medium may further include a culture additive such as glucose, glutamine, HAT and HT additives, and BSA (bovine serum albumin) according to the nature and culture purpose of the cultured cells, and may further include antibiotics or antifungal agents as necessary. have. The antibiotics include penicillin, streptomycin, gentamicin, neomycin, kanamycin, and the like. The antifungal agents include, but are not limited to, amphotericin B, nystatin, and the like.

The method of increasing the replication of norovirus of the present invention comprises inoculating norovirus into cells cultured as above and applying an electrical stimulus to the cells infected with the norovirus.

The inoculation of the norovirus may be directly inoculated with the purified norovirus to the cell, the sample contaminated with the norovirus, or the culture containing the cultured norovirus may be inoculated after filtering the microfilter.

As used herein, the term "electrical stimulation" means that a current is transmitted to a cell infected with a norovirus or a cell culture comprising the cell. The electrical stimulation is performed by applying pulses to two electrodes, and a negative electrode is called a 'cathode' and a positive electrode is called a 'anode'.

Specifically, the electrical stimulus is 0.1 to 1000 nA. More specifically, 0.1 to 800 nA, 0.1 to 500 nA, 0.1 to 300 nA, 0.1 to 200 nA, 0.2 to 1000 nA, 0.4 to 1000 nA, 0.6 to 1000 nA, 0.8 to 1000 nA. 0.2-800 nA, 0.4-5000 nA, 0.6-300 nA or 0.8-200 nA.

The electrical stimulus may be applied for a period of time.

Specifically, the electrical stimulus is applied for 1 to 10 days. More specifically, 1 to 9 days, 1 to 8 days, 1 to 7 days, 1 to 6 days, 1 to 5 days, 1 to 4 days, 2 to 10 days, 2 to 9 days, 2 to 8 days, Apply for 2-7 days, 2-6 days, 2-5 days, 2-4 days.

The electrical stimulation may be applied periodically for a predetermined time within the predetermined period.

Specifically, the electrical stimulation is applied for 10 to 360 minutes / day (day). More specifically, the electrical stimulation is 10 to 300 minutes / day, 10 to 240 minutes / day, 10 to 180 minutes / day, 10 to 120 minutes / day, 10 to 90 minutes / day, 20 to 360 minutes / day For 30 to 360 minutes / day, 20 to 300 minutes / day, 30 to 240 minutes / day, 30 to 180 minutes / day, 30 to 120 minutes / day, 30 to 90 minutes / day or 40 to 80 minutes / day Is authorized.

In the present invention, whether or not the replication of the norovirus can be confirmed by quantitative test of the virus, and the quantitative test of the virus can be used, such as plaque assay, fluorescent foci assay, pock assay, TCID ₅₀ method, but is not limited thereto. It is also possible to confirm by selecting a target gene from a marker protein or norovirus genome related to norovirus replication and measuring its expression level by qPCR.

We analyzed the expression level of norovirus by analyzing the expression levels of TFR and NCL involved in the replication of norovirus.

The transferrin receptor (TFR) is a major receptor for iron receptors entering cells and is a protein expressed in all tissues. The TFR is an arenavirus (arenaviruses), Machupo virus (Machupo virus), Junin virus (Junin virus), mouse mammary tumor virus (mouse mammary tumor virus (MMTV), canine parvovirus (canine parvovirus), feline leukopenia virus ( feline panleukopenia virus (FPV) has been identified as an entry receptor for many viruses.

The NCL (nucleolin) is a multifunctional protein that contributes extensively to DNA and RNA regulatory mechanisms, including ribosomal biogenesis, chromatin modification, mRNA stability and translation, nuclear export of RNA and protein complexes, and microRNA processing. to be. The NCL is involved in a variety of pathological processes, in particular viral infection, through its ability to bind various target RNAs.

According to another aspect of the invention, the present invention is a method for detecting norovirus, tuba4a (Tubulin Alpha 4a), tubulin (family), tubb1 (Tubulin Beta 1), tuba1b (Tubulin Alpha) of the cells infected with norovirus 1b), tubulin (complex), 19s proteasome, 19s proteasome, psmd2 (Proteasome 26S Subunit, Non-ATPase 2), alpha tubulin, eif2ak2 (Eukaryotic Translation Initiation Factor 2 Alpha Kinase 2), mx1 (MX Dynamin Like GTPase 1), fkbp4 (FK506 Binding Protein 4), gdi2 (GDP Dissociation Inhibitor 2), pnp (Purine Nucleoside Phosphorylase), pgd (Phosphogluconate Dehydrogenase), cct2 (Chaperonin Containing TCP1 Subunit 2), eef2 (eukaryotic translation) ), mdh2 (Malate Dehydrogenase 2), mthfd1 (Methylenetetrahydrofolate Dehydrogenase 1), hla-b (major histocompatibility complex, class I, B), lgals3bp (galectin 3 binding protein), hsp70 (heat shock protein 70), isg15 (Interferon- stimulated gene 15), hspa4 (Heat Shock Protein Family A (Hsp70) Member 4) , prdx1 (Peroxiredoxin 1), eif3a (Eukaryotic Translation Initiation Factor 3 Subunit A), tagln2 (Transgelin 2), rdx (Radixin), actn1 (Actinin Alpha 1), anxa2 (Annexin A2), apaf1 (Apoptotic Protease Activating Factor 1) , anxa1 (Annexin A1), eif4a3 (Eukaryotic Translation Initiation Factor 4A3), krt18 (Keratin 18), krt7 (Keratin 7), rpn1 (Ribophorin I), hspa5 (Heat Shock Protein Family A (Hsp70) Member 5), myh9 ( Myosin Heavy Chain 9), actn4 (Actinin Alpha 4), vim (Vimentin), cytokeratin, krt15 (keratin 15), hyou1 (hypoxia up-regulated 1), srek1 (Splicing Regulatory Glutamic Acid And Lysine Rich Protein 1), ncl (Nucleolin), mybbp1a (MYB Binding Protein 1a), hnrnpf (Heterogeneous Nuclear Ribonucleoprotein F) and des (Desmin), the method comprising detecting the expression level of one or more genes or proteins selected from the group consisting of To provide.

Since the detection method of the norovirus of the present invention uses the same norovirus, cell, and infection method as the method of increasing the replication of the norovirus, the common contents between the two are to avoid excessive complexity of the present specification. Omit the description.

Detection of the expression level of the gene or protein in the present invention can be carried out by various methods known in the art.

According to one embodiment of the invention, the gene expression level of step (b) may be carried out by a gene amplification method. In this case, the tuba4a, tubulin (family), tubb1, tuba1b, tubulin (complex), 19s proteasome, psmd2, alpha tubulin, eif2ak2, mx1, fkbp4, gdi2, pnp, pgd, cct2, eef2, mdh2 , mthfd1, hla-b, lgals3bp, hsp70, isg15, hspa4, prdx1, eif3a, tagln2, rdx, actn1, anxa2, apaf1, anxa1, eif4a3, krt18, krt7, rpn1, hspa5, myh9, actn4 keratin, vim It is carried out using a primer or probe that complementarily binds to a gene selected from the group consisting of krt15, hyou1, srek1, ncl, mybbp1a, hnrnpf and des.

As used herein, the term “primer” is complementary to the 5′-terminal or 3′-terminal sequence of the target nucleic acid site to be amplified in the nucleic acid amplification reaction, respectively, and is suitable for use in suitable buffers at suitable temperatures ( That is, it refers to a single-stranded oligonucleotide capable of acting as a starting point for the polymerase reaction of the template-directed nucleic acid under four different nucleoside triphosphates and polymerases.

The term "probe" as used herein is a single-chain nucleic acid molecule and includes sequences complementary to the target sequencing. According to one embodiment of the present invention, the probe of the present invention can be modified within the range that the advantages of the probe of the present invention, ie hybridization specificity, are not impaired. For example, a reporter phosphor or a quencher may be tagged at the end of the probe oligonucleotide.

In the real-time polymerase chain reaction of the present invention, the probe uses a probe capable of complementarily binding to some sequences inside the nucleotide sequence amplified by the primer pair.

According to a preferred embodiment of the present invention, the reporter-phosphor or quencher is tagged at the 5'-end and 3'-end of the probe used in the real-time polymerase chain reaction (PCR) of the present invention. Can be.

Various DNA polymerases may be used in the amplification reaction in the method of the present invention, and preferably, the DNA polymerase is a thermostable DNA polymerase obtainable from various bacterial species. This includes Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis, Thermis flavus, Thermococcus literalis, and Pyrococcus furiosus (Pfu).

On the other hand, when performing a gene amplification reaction, it is preferable to provide an excess amount of components necessary for the reaction to the reaction vessel. Excess of components required for the amplification reaction means an amount such that the amplification reaction is not substantially limited to the concentration of the components. So that the degree of amplification to a desired joinja, dATP, dCTP, dGTP and dTTP, such as Mg ^{2 +} can be achieved to provide the reaction mixture is required. All enzymes used in the amplification reaction may be active under the same reaction conditions. In fact, the buffer ensures that all enzymes are close to optimal reaction conditions. Thus, the amplification process of the present invention can be carried out in a single reactant without changing conditions such as addition of the reactants.

According to another preferred embodiment of the invention, the kit further comprises a buffer, a DNA polymerase, a DNA polymerase cofactor and dNTPs.

According to another embodiment of the invention, the gene or protein expression level of step (b) may be carried out by an antigen-antibody reaction mode. In this case, the tuba4a, tubulin (family), tubb1, tuba1b, tubulin (complex), 19s proteasome, psmd2, alpha tubulin, eif2ak2, mx1, fkbp4, gdi2, pnp, pgd, cct2, eef2, mdh2 , mthfd1, hla-b, lgals3bp, hsp70, isg15, hspa4, prdx1, eif3a, tagln2, rdx, actn1, anxa2, apaf1, anxa1, eif4a3, krt18, krt7, rpn1, hspa5, myh9, actn4 keratin, vim It is carried out using an antibody or aptamer that specifically binds to a gene or protein selected from the group consisting of krt15, hyou1, srek1, ncl, mybbp1a, hnrnpf and des.

As used herein, the term “antibody” refers to a specific protein molecule directed against an antigenic site. For the purposes of the present invention, antibodies are markers of the invention (tuba4a, tubulin (family), tubb1, tuba1b, tubulin (complex), 19s proteasome, psmd2, alpha tubulin, eif2ak2, mx1, fkbp4, gdi2, pnp, pgd, cct2, eef2, mdh2, mthfd1, hla-b, lgals3bp, hsp70, isg15, hspa4, prdx1, eif3a, tagln2, rdx, actn1, anxa2, apaf1, anxa1, eif4a3, krt18, krt7, rpn myh9, actn4, vim, cytokeratin, krt15, hyou1, srek1, ncl, mybbp1a, hnrnpf or des) or an antibody that specifically binds to the constituent proteins of the markers, including polyclonal, monoclonal and recombinant antibodies Includes all of them. The antigen-antibody reaction method has the same meaning as the immunoassay method.

The polyclonal antibody can be produced by methods well known in the art for injecting the marker protein antigen described above into an animal and collecting blood from the animal to obtain a serum comprising the antibody. Such polyclonal antibodies can be prepared from any animal species host such as goat, rabbit, sheep, monkey, horse, pig, bovine dog.

Such monoclonal antibodies are known in the art by the hybridoma method (Kohler and Milstein, European Jounral of Immunology 6: 511-519, 1976) or phage antibody libraries (Clackson et al. Nature, 352: 624). -628, 1991; Marks et al. J. Mol. Biol., 222: 58, 1-597, 1991).

Antibodies prepared by the above method can be isolated and purified using methods such as gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, affinity chromatography, and the like. In addition, antibodies of the invention include functional fragments of antibody molecules, as well as complete forms having two full length light chains and two full length heavy chains. A functional fragment of an antibody molecule refers to a fragment having at least antigen binding function, and includes Fab, F (ab '), F (ab') 2 and Fv.

When the method of the present invention is carried out using antibodies or aptamers, the present invention can be used to detect noroviruses according to conventional immunoassay methods.

This immunoassay can be used to detect norovirus by performing according to various quantitative or qualitative immunoassay methods developed in the prior art. The immunoassay format is Western blot, enzyme linked immunosorbent assay (ELISA), capture-Eliza, radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket ) Immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, Fluorescence Activated Cell Sorter (FACS), protein chip, etc. It does not limit the analysis method of the present invention. The immunoassay or method of immunostaining is described in Enzyme Immunoassay, E. T. Maggio, ed., CRC Press, Boca Raton, Florida, 1980; Gaastra, W., Enzymelinked immunosorbent assay (ELISA), in Methods in Molecular Biology, Vol. 1, Walker, J.M. ed., Humana Press, NJ, 1984; And Ed Harlow and David Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999, which is incorporated herein by reference.

The method for detecting norovirus of the present invention can detect norovirus by analyzing the expression level of the marker.

Specifically, the Tuba4a, tuba1b, 19s proteasome, psmd2, alpha tubulin, mx1, fkbp4, gdi2, pgd, eef2, mdh2, mthfd1, lgals3bp, hsp70, hspa4, prdx1, eif3a, rdx, actn1, anxa2, anxa3, anxa2, anxa determined to be infected with norovirus when one or more genes or proteins selected from the group consisting of krt18, krt7, rpn1, hspa5, myh9, actn4, vim, hyou1, srek1, ncl, mybbp1a, hnrnpf and des are up-regulated do.

Specifically, at least one gene or protein selected from the group consisting of tubulin (family), tubulin (complex), tubb1, eif2ak2, cct2, pnp, isg15, hla-b, apaf1, tagln2, krt15, and cytokeratin is downward. If controlled, it is determined to be infected with norovirus.

The term “up-regulation” or “down-regulation” as used herein referring to a gene or protein refers to the expression of a gene or protein in a sample in which the degree of expression of the gene or protein in the sample under investigation does not infect the norovirus. Compared with degree, it means 'high' or 'low' respectively.

In determining norovirus infection of the present invention, the up-regulation means that the amount of expression is increased by at least 1.1 times, at least 1.3 times or at least 1.5 times. The down-regulation means a decrease in expression level of 0.9 times or less, 0.7 times or less, or 0.5 times or less.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a method for increasing replication of norovirus and a method for detecting norovirus.

(b) The present invention enables the study of infection and disease of norovirus by culturing and propagating the norovirus in a laboratory.

1 is a device for providing electrical stimulation to culture cells and a device for controlling the same.
Figure 2 shows the results of protein body analysis of zebrafish infected with human-type norovirus.
Figure 3 shows the results of protein body analysis in MDCK host cells infected with human-type norovirus. Red protein is increased protein, and green protein means decreased protein. Colored solid lines highlight the interactions between proteins involved in the viral infection mechanism.
Figure 4 shows the effect of increasing the expression of human infection-type norovirus replication-related proteins TFRC and NCL when subjected to electrical stimulation.
Figure 5 shows the effect of increasing the replication of human infectious norovirus when treated with electrical stimulation.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Throughout this specification, "%" used to indicate the concentration of a particular substance is (weight / weight)% solids / solids, (weight / volume)%, unless otherwise indicated, and Liquid / liquid is (volume / volume)%.

Example  1. Cell Culture

MDCK cells (ATCC CCL-34) were cultured in a 37 ° C. incubator with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin in DMEM medium.

Example  2. Virus Inoculation

MDCK cells were infected with human-type norovirus GII-4 with a multiplicity of infection (MOI) 0.05 and cultured in a 30 ° C. incubator for 72 hours.

Example  3. Application of Electrical Stimulation

Electric stimulation of 1 nA or 100 nA was applied for 1 hour after 24 hours of incubation of MDCK cells in a 37 ° C. incubator. After 24 hours after the electrical stimulation, the human norovirus was infected, and 1 nA or 100 nA was applied to the 30 ° C. incubator at 0, 24 and 48 hpi (hour post infection (hpi)) for 1 hour.

Example  4. PCR  through Norovirus  Confirmation of Replication-Related Protein Expression

In this embodiment, in order to confirm the expression of norovirus replication-related proteins using electrical stimulation, 1 nA or 100 nA electrical stimulation was performed for 12 hours after the culture of the cells, RNA was extracted from the cells, and PCR was performed.

Human infectious norovirus replication-related proteins include the transferrin receptor according to the Norovirus Proteomics results (Publication Publication No. 10-2017-0007000 (2017.01.16) "Natural composition for neutralizing human infectious norovirus"). (Transferrin Receptor (TFR) and Nucleolin (NCL) expression was confirmed.

As can be seen in Figure 2, since the expression of TFRC also appears in human norovirus protein body analysis results, it was selected as a replication-related protein.

As shown in FIG. 3, the expression of NCL also increases in proteomic analysis of cells infected with human-infected norovirus, and thus was selected as a replication-related protein. In FIG. 3, Hsp70, ANXA2, and NCL were overexpressed when infected with human type norovirus infected with MDCK cells, and based on this, NCL protein was less than 100 nA under optimal conditions for human type norovirus replication in FIG. 4. Overexpression was confirmed.

RNA was isolated from cells using TRIzol (Invitrogen) and performed according to a general RNA preparation protocol (Chomczynski P, Mackey K. Short technical report.Modification of the TRIzol reagent procedure for isolation of RNA from Polysaccharide-and proteoglycan-rich sources) Biotechniques 19 (6): 942-945. 1995). 700 μl of Trizol was added to a 1.5 ml tube and lightly vortexed, and then 200 μl of chloroform was vortexed and allowed to stand for 5 minutes. The supernatant was removed by centrifugation at 4 ° C. for 10 minutes at 12,000 × g, and 500 μl of 75% ethanol was dispensed and gently vortexed. After centrifugation at 7,500 × g, 5 min, 4 ° C., the supernatant was removed and RNA was obtained using 10 μl of RNase-free ultrapure water (RNase free water). 10 μl of the obtained RNA was prepared by dispensing with 1 μl of a random primer and reacting at 65 ° C. for 10 minutes, at 37 ° C. for 1 hour, and at 72 ° C. for 5 minutes. 10 μl of SYBR for probes, 7 μl of free water, 0.5 μl of probe, 0.5 μl of forward primer and reverse primer were added, and 2 μl of cDNA synthesized were reacted together to perform qRT-PCR. qRT-PCR conditions are 45 cycles 95 degreeC 5 second, 55 degreeC 10 second, 72 degreeC 20 second. Primer and probe sequences for norovirus detection are as follows:

Probe or Primer Name order Sequence Listing RING2P (probe) TGGGAGGGCGATCGCAATCT First sequence JJV2F (Forward Primer) CAAGAGTCAATGTTTAGGTGGATGAG 2nd sequence COG2R (Reverse Primer) TCGACGCCATCTTCATTCAC Third Sequence

Figure 4 shows the results of comparing the TFR and NCL expression levels of the experimental group 1 nA and 100 nA to which the control (Mock) and the electrical stimulation did not apply the electrical stimulation to 1 nA and 100 nA, respectively. Expression of TFR and NCL, which are proteins related to norovirus replication, was increased when electrical stimulation was applied to cells. When 1 nA was compared with 100 nA, high expression of proteins was confirmed at 1 nA.

Example  5. PCR  through Norovirus  Identify the effect of increased replication

MDCK cells infected with human infectious norovirus (Genotype GII-4) were subjected to electrical stimulation of 1 nA or 100 nA for 1 hour for 4 days (1 day before infection and 3 days after infection). RNA was obtained from the supernatant 72 hours after infection of the cells with human infectious norovirus. RNA isolation was performed using TRIzol (Invitrogen) and performed according to a general RNA preparation protocol (Chomczynski P, Mackey K. Short technical report.Modification of the TRIzol reagent procedure for isolation of RNA from Polysaccharide-and proteoglycan-rich sources.Biotechniques 19 (6): 942-945. 1995). 700 μl of Trizol was added to a 1.5 ml tube and lightly vortexed. Then, 200 μl of chloroform was dispensed by vortexing and allowed to stand for 5 minutes. The supernatant was removed by centrifugation at 4 ° C. for 10 minutes at 12,000 × g, and 500 μl of 75% ethanol was dispensed and gently vortexed. After centrifugation at 7,500 × g, 5 min, 4 ° C., the supernatant was removed and RNA was obtained using 10 μl of RNase-free ultrapure water (RNase free water). 10 μl of the obtained RNA was prepared by dispensing with 1 μl of a random primer and reacting at 65 ° C. for 10 minutes, at 37 ° C. for 1 hour, and at 72 ° C. for 5 minutes. 10 μl of SYBR for probes, 7 μl of pure water, 0.5 μl of probe, 0.5 μl of forward primer and reverse primer were added, and 2 μl of cDNA synthesized were reacted together to perform qRT-PCR. qRT-PCR conditions are 45 cycles 95 degreeC 5 second, 55 degreeC 10 second, 72 degreeC 20 second. Primer and probe sequences are shown in Table 1.

5 is a control group Mock not infected with human infectious norovirus, experimental group NV infected with human infectious norovirus only without electrical stimulation, experimental group NV 1 nA, norovirus infected and 1 nA applied electrical stimulation, noro Norovirus concentrations of experimental group NV 100 nA with virus infection and 100 nA electrical stimulation are shown. Comparing the NV, NV 1 nA and NV 100 nA virus treatment groups, it was confirmed that the replication of human infectious norovirus (Genotype GII-4) was increased in cells treated with current conditions compared to NV without electric stimulation. In the case of applying 1 nA of electrical stimulation, the infection rate of human norovirus was highest.

The results of FIG. 5 were calculated by substituting the ct value of qRT-PCR as the x value in the formula y = -3.498x + 42.305 and R ² = 0.9974 calculated from the norovirus standard curve to calculate the norovirus quantitative value.

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that such a specific technology is only a preferred embodiment, and the scope of the present invention is not limited thereto. Therefore, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

<110> INDUSTRY FOUNDATION OF CHONNAM NATIONAL UNIVERSITY <120> Method for Promoting Norovirus Replication <130> PN170124 <160> 3 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> RING2P <400> 1 tgggagggcg atcgcaatct 20 <210> 2 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> JJV2F <400> 2 caagagtcaa tgtttaggtg gatgag 26 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> COG2R <400> 3 tcgacgccat cttcattcac 20

Claims (11)

In a method of increasing the replication of norovirus,
A method comprising the step of applying an electrical stimulation of 0.1 to 1000 nA to cells other than cells in the human body infected with the norovirus.
The method of claim 1, wherein the norovirus is a norovirus whose genotype is GI, GII, or GIV.
The method of claim 1, wherein the norovirus is genotype is GII-4 (Genotype GII-4) characterized in that the method.
The method of claim 1, wherein the cells are Madin Darby canine kidney (MDCK), Madin Darby bovine kidney (MDBK), Caco-2, Ht-29, HuTu-80, Detroit 551 ( Detroit 551), Detroit 562, HTC-8, Microbiological Associates 104 (MA104), AGS, I-407, Vero, HEC, Cotton Rat-Primary Epithelial Cells (Cotton rat) primary epithelial cells (CR-PEC), fetal rhesus monkey kidney (FRhK-4), HeLa, Kato-3, Crandell Rees Feline Kidney (CRFK) and IEC- At least one cell selected from the group consisting of 6.
delete The method of claim 1 wherein the electrical stimulus is applied for 1 to 10 days.
The method of claim 1 wherein the electrical stimulus is applied for 10 to 360 minutes / day.
In the infection detection method of norovirus,
Tubulin alpha 4a (tuba4a), tubulin family, tubulin beta 1 (tubb1), tubulin alpha 1b (Tubulin Alpha 1b; tuba1b), tubulin complex, 19s proteasome, proteasome 26s subunit non-ATPase 2 (psmd2), alpha tubulin, eukaryotic translation initiation factor 2 alpha kinase 2 ( Eukaryotic Translation Initiation Factor 2 Alpha Kinase 2; eif2ak2), MX Dynamin Like GTPase 1; mx1, FK506 Binding Protein 4; fkbp4, GDP Dissociation Inhibitor 2 2; gdi2), Purine Nucleoside Phosphorylase (pnp), Phosphogluconate Dehydrogenase (pgd), Chaperronin Containing TCP1 Subunit 2; cct2 Eukaryotic translation kidney phosphorus) 2 (Eukaryotic Translation Elongation Factor 2; eef2), Malate Dehydrogenase 2 (mdh2), Methylenetetrahydrofolate Dehydrogenase 1 (mthfd1), Major histocompatibility complex, class I, B; hla-b), galectin 3 binding protein (lgals3bp), heat shock protein 70; hsp70), Interferon-stimulated gene 15; isg15, Heat Shock Protein Family A (Hsp70) Member 4; hspa4), peroxredoxin 1 (Peroxiredoxin 1; prdx1), Eukaryotic Translation Initiation Factor 3 Subunit A; eif3a, Transgelin 2 (tagln2), Radixin (rdx), Actinin alpha 1 (Actinin Alpha 1; actn1), Annexin A2; anxa2, Apoptotic Protease Activating Factor 1 (apap1), Annexin A1 (Annexin A1; anxa1), Eukaryotic Translation Initiation Factor 4A3 (Eukaryotic Translation Initiation Factor 4A3; eif4a3), Keratin 18 (krt18), Keratin 7 (krt7), Ribophorin 1 (rpn1), Heat Shock Protein Family A (Hsp 70) Member 5 ( Heat Shock Protein Family A (Hsp70) Member 5; hspa5), Myosin Heavy Chain 9; myh9, Actinin Alpha 4; actn4), vimentin (vim), cytokeratin, keratin 15 (keratin 15; krt15), hypoxia up-regulated 1 (hyou1), splicing regulating glutamine / lysine-rich protein 1 (Splicing regulatory glutamine / lysine-rich protein 1; srek1), nucleolin (ncl), MYB binding protein 1a (MYB Binding Protein 1a; mybbp1a), detecting the expression level of one or more genes or proteins selected from the group consisting of heterogeneous Nuclear Ribonucleoprotein F (hnrnpf) and Desmin (des). As a method,
The tubulin alpha 4a, tubulin alpha 1b, 19s proteasome, proteasome 26s subunit non-etipiase 2, alpha tubulin, MX dynamin-like getipiaase 1, FK506 binding protein 4, GDP dissociation inhibitor 2, Phosphogluconate dehydrogenase, eukaryotic translation elongation factor 2, malic dehydrogenase 2, methylenetetrahydrofolate dehydrogenase, galectin 3 binding protein, heat shock protein 70, heat shock protein family A (Hsp 70) member 4, ph. Roxyredoxin 1, eukaryotic translation initiation factor 3 subunit A, radicin, actinin alpha 1, annexin A2, annexin A1, eukaryotic translation initiation factor 4A3, keratin 18, keratin 7, ribophorin 1, heat shock Protein family A (Hsp 70) member 5, myosin heavy chain 9, actinin alpha 4, bimentin, hypoxia up-regulated 1, splicing regulatory glutamine / lysine-rich protein 1, nucleoline, MYB binding protein 1a, Heterologous Nuclei Nucleoprotein F and Death When the expression level of the NM gene or protein is high compared to the expression level of the gene or protein of a cell which has not infected the norovirus, it is determined that the infection is caused by the norovirus;
The tubulin family, tubulin complex, tubulin beta 1, eukaryotic translation initiation factor 2 alpha kinase 2, chaperonin containing TCP1 subunit 2, purine nucleoside phosphorylase, interferon-stimulated gene 15, main If the expression level of histocompatibility complex class 1-B, apoptotic protease activator 1, transgelin 2, keratin 15 and cytokeratin gene or protein is low compared to the gene or protein expression level of cells not infected with norovirus, Determining to be infected with a virus.
The method of claim 8, wherein the norovirus is a norovirus whose genotype is GI, GII, or GIV.
9. The method of claim 8, wherein the norovirus is genotype GII-4 (Genotype GII-4).
The method of claim 8, wherein the cells are Madin Darby canine kidney (MDCK), Madin Darby bovine kidney (MDBK), Caco-2, Ht-29, HuTu-80, Detroit 551 ( Detroit 551), Detroit 562, HTC-8, Microbiological Associates 104 (MA104), AGS, I-407, Vero, HEC, Cotton Rat-Primary Epithelial Cells (Cotton rat) primary epithelial cells (CR-PEC), fetal rhesus monkey kidney (FRhK-4), HeLa, Kato-3, Crandell Rees Feline Kidney (CRFK) and IEC- At least one cell selected from the group consisting of 6.

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