KR20220018919A - Pharmaceutical composition for the prevention and treatment of Envelopled viruses comprising taining Platycodon grandiflorus root extracts - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for the prevention and treatment of COVID-19 containing a Platycodon grandiflorum extract and platycodin D as active components. More specifically, the present invention relates to a pharmaceutical composition for prevention and treatment of COVID-19 by inhibiting cellular infection of COVID-19 and inhibiting the production of intracellular infectious virus of platicodin D, a component contained in Platycodon grandiflorum, and a method for preventing and treating COVID-19 by using the pharmaceutical composition. The Platycodon grandiflorum extract or platycodin D, an active component thereof exhibits a remarkable inhibitory effect on cell entry and cell infection of coronavirus, and is confirmed to exhibit a more pronounced effect than chloroquine, camostat, and nafamostat, which are treatment candidates through inhibition of cell entry and cell infection of coronavirus. Through the same, the pharmaceutical composition can be usefully used to prevent coronavirus or to treat early stages of infection. In addition, the Platycodon grandiflorum extract or platycodin D inhibits the cleavage of the spike protein important for infection during packaging of coronavirus, thereby inhibiting the proliferation and spread of the infectious coronavirus in cells. Therefore, the composition of the present invention is useful for the prevention and treatment of coronavirus.

Description

Pharmaceutical composition for the prevention and treatment of enveloped viruses including Gilgyeong extract

The present invention relates to a pharmaceutical composition for the prevention and treatment of COVID-19 containing Gilgyeong extract, and/or Platycodin D as an active ingredient, and more particularly, a component contained in Gilkyung. It relates to a pharmaceutical composition for the prevention and treatment of COVID-19 through inhibition of cell infection and inhibition of production of intracellular infectious virus for coronavirus 19 of Platicodin D, and a method for preventing and treating COVID-19 using the pharmaceutical composition .

Coronavirus (Corona Virus) is a type of RNA virus, a virus that consists of ribonucleic acid (RNA) genetic information, and causes infection of the respiratory and digestive systems of humans and animals. Corona virus can be easily transmitted mainly through mucosal transmission and droplet transmission, and it usually causes mild respiratory infections in humans, but rarely causes fatal infections that lead to death.

Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), a kind of the above coronavirus, emerged from Wuhan, China and spread rapidly around the world, and the World Health Organization (WHO) declared a disease infected with the virus as COVID- 19 was named. According to a recent report, common symptoms of infection include fever (98.6%), fatigue (69.6%), dry cough (59.4%), lymphopenia (70.3%), prolonged prothrombin time (58%) and increased lactate dehydrogenase. (39.9%) and the like (Wang, D. et al., JAMA, 2000).

It has been reported that the SARS-CoV-2 is mainly transmitted through human-to-human contact through respiratory droplets formed by coughing and sneezing, and the surface of objects contaminated by coughing or sneezing (CDC, Cfdcap, How COVID- 19 Spreads, Coronavirus Disease 2019 (COVID-19), 2020), even asymptomatic infection has been reported to be possible (Yu, P., et al., J Infect Dis, 2020; Hoehl, S., et al., N Engl J Med, 2020; Bendix A., Science Alert, 2020)

As SARS-CoV-2 cases surge around the world, WHO declared a 'Public Health Emergency of International Concern' (PHEIC) on January 30, 2020, and as the number of confirmed cases of coronavirus infection spread across the world, WHO announced in March On the 11th, after Hong Kong Flu (1968) and H1N1 Flu (2009), it was the third time in history that COVID-19 was declared a pandemic.

Currently, pharmaceutical companies in several countries, including the United States and the United Kingdom, have developed vaccines and therapeutics for the prevention or treatment of SARS-CoV-2, and some of them are approved in some countries and administered to the general public. However, in the case of the vaccine, there are a number of problems due to the use of a vaccine that has not undergone a complete clinical procedure, such as causing severe side effects or, in severe cases, death of a person receiving the vaccine, and due to the emergence and spread of mutant viruses, Controversy arises over the efficacy of vaccines.

Therefore, the clinical effect is uniformly excellent regardless of the patient's characteristics, immunity, condition, etc., and the therapeutic effect is excellent, especially for patients with underlying diseases, or the elderly with weak immunity, including young children and the elderly, COVID- 19 There is an urgent need to develop a therapeutic agent that is specific and has clinically proven therapeutic effects.

Giulio N. et al., Eur J. Pharmacol. 882:173328 (2020.6.27.)

As a result of efforts made by the present inventors to solve the above problems, platycodin D, an active ingredient contained in Gilkyung extract, enhances organ immunity known in relation to respiratory diseases, various anti-inflammation related to bronchus, and drainage In addition to the effect, two major pathways of entry into coronavirus cells were i) lysosomal cathepsins and ii) inhibitory ability on coronavirus-cell membrane fusion following cleavage of the coronavirus spike protein by TMPRSS2 in the cell membrane. In addition, it was confirmed that Platicodin D inhibits the cleavage of the SARS-CoV-2 spike protein in the viral packaging process, thereby preventing viral recombination and release, thereby inhibiting the production of infectious virus in cells, thereby completing the present invention. reached

That is, an object of the present invention is a composition containing platycodin D as an active ingredient or a gilgyeong root extract that inhibits cell entry or cell infection of corona virus and corona virus infection-19 (COVID-19) using the same ) to provide a method of prevention or treatment.

The pharmaceutical composition for preventing or treating severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection according to the present invention contains Platycodin D as an active ingredient.

In the pharmaceutical composition for preventing or treating severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection according to an embodiment of the present invention, the Platycodin D (Platycodin D) is the SARS-CoV-2 It may be to inhibit cell entry and/or inhibit binding to SARS-CoV-2 spike protein.

In the pharmaceutical composition for preventing or treating severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection according to an embodiment of the present invention, the Platycodin D (Platycodin D) is 0.001 based on the total weight of the composition to 50% by weight may be included.

A pharmaceutical composition for preventing or treating severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) according to the present invention includes an extract of Gilkyung ( Platycodon grandiflorum ).

In the pharmaceutical composition for preventing or treating severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection according to an embodiment of the present invention, the Gilkyung ( Platycodon grandiflorum ) extract enters the cells of SARS-CoV-2 Inhibitory and/or inhibiting the binding to the SARS-CoV-2 spike protein may be.

In the pharmaceutical composition for preventing or treating severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection according to an embodiment of the present invention, the gilgyeong ( Platycodon grandiflorum ) extract is 0.001 to 50 based on the total weight of the composition It may be included in weight %.

The present invention provides a pharmaceutical preparation comprising a pharmaceutical composition for preventing or treating the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection.

In the pharmaceutical formulation according to an embodiment of the present invention, the pharmaceutical formulation may be a solid dosage form or a liquid dosage form.

In the pharmaceutical preparation according to an embodiment of the present invention, the pharmaceutical preparation may be for oral, parenteral, nasal, oral, oral, sublingual, airway spray or rectal administration.

In the pharmaceutical formulation according to an embodiment of the present invention, the pharmaceutical formulation may be in the form of capsules, pills, tablets, solutions, suspensions, sprays, foams, patches or pastes.

The food composition for preventing or improving severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection according to the present invention contains Gilkyung ( Platycodon grandiflorum ) extract and/or Platycodin D (Platycodin D).

In the food composition for preventing or improving severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection according to an embodiment of the present invention, the Gilgyeong ( Platycodon grandiflorum ) extract, Platycodin D (Platycodin D) Or the mixture may be included in an amount of 0.001 to 50% by weight based on the total weight of the composition.

The method for preventing or treating severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection according to the present invention comprises the steps of administering an extract and / or Platycodin D ( Platycodin D) to a subject includes

In the method for preventing or treating severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection according to the present invention, the administration is oral, parenteral, nasal, oral, oral, sublingual, airway spray or rectal. it may be through

In the method for preventing or treating severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection according to the present invention, when the administration is performed by airway spray, the Platycodon grandiflorum extract and/or Platycodon Tycodin D (Platycodin D) may be applied to the respiratory tract to show the effect of preventing SARS-CoV-2 infection.

In the method for preventing or treating severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection according to the present invention, when performed by airway spray, the gilgyeong ( Platycodon grandiflorum ) extract, Platycodin D (Platycodin) D) or a mixture thereof may be included in an amount of 1 to 20% by weight based on the total weight of the composition.

In the method for preventing or treating severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection according to the present invention, the gilgyeong ( Platycodon grandiflorum ) extract may be in the form of granules or powder.

In the method for preventing or treating severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection according to the present invention, the gilgyeong ( Platycodon grandiflorum ) extract may be in the form of granules or powder for airway injection.

The Gilkyung extract of the present invention or its active ingredient, Platicodin D, exhibits a remarkable inhibitory effect on the cell entry and cell infection of the coronavirus, and chloroquine (Chloroquine ), remdesivir (Remdesivir), camostat (Camostat), it was confirmed that shows a more pronounced effect than nafamostat (Nafamostat). Through this, it can be usefully used to prevent corona virus or to treat early stages of infection. In addition, the Gilkyung extract or Platicodin D inhibits the cleavage of the spike protein important for infection during packaging of coronavirus, thereby inhibiting the proliferation and spread of the infectious corona virus in cells. Therefore, the composition of the present invention is useful for the prevention and treatment of coronavirus.

1 schematically shows the preparation of a pseudovirus expressing a lentivirus-based coronavirus spike protein according to the present invention and infection of parental H1299 cells stably expressing ACE2, a coronavirus receptor.
Fig. 2 shows the expression of fluorescent protein in ACE2-expressing H1299 cells transduced with SARS-CoV-2 spike-like lentiviral particles containing Green Fluorescent Protein ( GFP ) and firefly luciferase gene (Fig. 2a) and luciferase. This is the result of confirming the bioluminescence activity of the enzyme (Fig. 2b).
3 is a result confirming the inhibitory effect of platycodin D (platycodin D) on the entry into cells of the SARS-CoV-2 pseudovirus through the expression level of green fluorescent protein.
4 is a result of analyzing the effect of plasmodin D on inhibiting entry into cells of SARS-CoV-2 pseudovirus without cytotoxicity through luciferase bioluminescence.
5 is a result showing that saponin contained in ginseng has no effect of inhibiting entry into SARS-CoV-2 cells.
Figure 6 shows that plasmodin D is a treatment candidate through the inhibition of the existing cell entry and cell infection for both corona virus entry through lysosome by ACE2 single expression or TMPRSS by ACE2/TMPRSS2 co-expression. It is the result of inhibiting phosphorus more effectively than chloroquine, camostat, and nafamostat.
7 is a result confirming the inhibitory effect of a natural drug (Ryongaksan) containing gilgyeong root powder to inhibit the entry into cells of SARS-CoV-2 pseudovirus (FIG. 7a). This is the result of confirming the inhibitory effect of SARS-CoV-2 pseudovirus in cell entry with only a short period of pre-treatment with Yonggaksan (Fig. 7b). This is the result of confirming that the components of licorice powder contained in Yonggaksan do not have the effect of inhibiting the entry of SARS-CoV-2 pseudovirus into cells (FIG. 7c).
8 is a result confirming the inhibitory effect of SARS-CoV-2 pseudovirus in the cell entry of food made from gilgyeong (bellflower green).
9 is a result of confirming through liquid chromatography (HPLC) analysis, etc., that only platycodin D (platycodin D) exhibits the effect of inhibiting the entry into cells of SARS-CoV-2 in natural medicines and foods made with ryukaksan and gilgyeong; to be
10 is an analysis result of the ACE2 binding inhibitory ability of platicodin D to the SARS-CoV-2 spike protein receptor binding domain (RBD).
11 is a result confirming the inhibitory effect of platicodin D on spike protein cleavage during SARS-CoV-2 virus packaging.
12 is a result of analyzing live infectious SARS-CoV-2 virus and dose-response curves by immunofluorescence assay.
13 is a result of staining that Platincodin D changes the location and amount of cholesterol in the cell (FIG. 13a) and shows the fact that the change in cholesterol in the cell plays an important role in inhibiting the entry of coronavirus (FIG. 13b) ).
14 is a result confirming through modeling that platincodin D forms a complex with methyl-beta-cyclodextrin (MβCD) with higher affinity than cholesterol.
15 is a result of confirming that Platicodin D can be placed inside the cell membrane together with cholesterol through molecular modeling.
16 is a result confirming that Platincodin D inhibits cell membrane fusion.
17 is a result of platycodin D inhibiting the entry of SARS-CoV-2 pseudovirus through autophagy in different types of cells.
18 schematically shows the mechanism by which platincodin D inhibits the intracellular entry of SARS-CoV-2 by interfering with virus-intercellular membrane fusion in ACE+ and ACE2/TMPRSS2+ cells.
19 schematically shows the location of the action of existing research and development drugs in the mechanism of coronavirus cell infection and the ability of Platicodin D of the present invention to inhibit cell entry and production of cell-infectious viruses.

Hereinafter, with reference to the accompanying tables or drawings, a pharmaceutical composition for the prevention and treatment of COVID-19 comprising the gilgyeong extract of the present invention as an active ingredient will be described in detail.

When drawings are described, they are provided as examples so that the spirit of the present invention can be sufficiently conveyed to those skilled in the art. Therefore, the present invention is not limited to the drawings presented and may be embodied in other forms, and the drawings may be exaggerated to clarify the spirit of the present invention.

At this time, if there is no other definition in the technical terms and scientific terms used, it has the meaning commonly understood by those of ordinary skill in the art to which this invention belongs, and the gist of the present invention in the following description and accompanying drawings Descriptions of known functions and configurations that may be unnecessarily obscure will be omitted.

Also, the singular form used in the specification of the present invention may also be intended to include the plural form unless the context specifically dictates otherwise.

In addition, in the specification of the present invention, the unit used without special mention is based on the weight, for example, the unit of % or ratio means weight % or weight ratio.

In addition, in the specification of the present invention, the expression “comprising” is an open-ended description having an equivalent meaning to expressions such as “comprising”, “containing”, “having” or “characterized by”, and additionally listed It does not exclude elements, materials or processes that do not exist. Also, “really… The expression “consisting of” means that other elements, materials or processes not listed together with the specified elements, materials or processes do not unacceptably significantly affect at least one basic and novel technical idea of the invention. It means that it can exist in quantity. Also, the expression “consisting of” means that only the elements, materials, or processes described are present.

As used herein, the terms "ingredient", "composition", "composition of a compound", "compound", "drug", "pharmaceutically active agent", "active agent", "healing" "therapy" "treatment" or "agent" is used interchangeably to mean a compound or composition of compound(s) or substances that, when administered to a subject (human or animal), induces a desired pharmaceutical and/or physiological effect by local and/or systemic action. used interchangeably.

As used herein, the term “treatment” or “therapy” (as well as its different forms) includes prophylactic (eg, prophylactic treatment), curative or palliative treatment. As used herein, the term “treating” includes alleviating or reducing at least one adverse or adverse effect or symptom of a condition, disease or disorder. The terms "prevention", "improvement" and "treatment" of the present invention should be interpreted in the broadest sense, and "prevention" refers to a patient who may be exposed to a disease or susceptible to a disease, but has not yet experienced or revealed symptoms of the disease. It means that one or more of the clinical symptoms of the disease do not progress in the patient. "Treatment" means any action that arrests or reduces the development of a disease or one or more clinical symptoms thereof.

In the present invention, “sample” or “sample” refers to an object for analysis, and is used in the same sense throughout the specification.

In the present invention, “platycodin D” is used interchangeably with the same meaning as “PD” throughout the specification.

Hereinafter, a pharmaceutical composition for preventing or treating severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection according to the present invention will be described in detail.

The pharmaceutical composition for preventing or treating severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection according to the present invention contains Platycodin D as an active ingredient.

Gilgyeong (桔梗, bellflower, Platycodon grandiflorus ) is a traditional edible medicinal herb widely used in Korea, China and Japan. Gilkyung is rich in various compounds such as saponin, flavonoids, and phenolic acid, and is known to have effects such as hypotension, lipid reduction, atherosclerosis, inflammatory cough and phlegm relief. In particular, it is known to be effective in drainage of tuberculosis, seawater, and sore throat. Due to the above effects, gilkyung has been used for sore throat, cough due to cold, phlegm, nasal congestion, asthma, bronchial inflammation, pleurisy, headache, chills, tonsillitis, and the like.

Triterpenoid saponins are a group of saponin components abundant in Gilgyeong ( Platycodon grandiflorus ). Currently, 75 triterpenoid glycosides have been isolated and identified from Gilgyeong.

Among them, platycodin A is considered to be the main saponin component of Gilkyung. Pladicodin D is known to have anti-inflammatory, anti-obesity, cholesterol lowering, blood sugar lowering effects, and the like, and is known to be related to immunity enhancement. However, the antiviral effect of gilkyung extract, particularly platyconin D (PD), in particular, the antiviral effect specific to the coronavirus has not been revealed at all.

The present invention provides an effect of inhibiting the entry of corona virus into cells of the gilgyeong root extract, particularly Platicodin D, which is abundantly contained in the root and root bark of gilgyeong, and the effect of inhibiting the cleavage of the coronavirus spike protein, which is important for infection, in the viral packaging stage. It is characterized by discovering for the first time and applying it for the purpose of preventing or treating coronavirus. In particular, in one embodiment of the present invention, while Platicodin D does not inhibit the entry of other viruses, vesicular stomatitis virus (VSV), it inhibits the interaction of the corona virus spike protein and its receptor, so that the coronavirus-specific cells It was newly confirmed that it has an entry inhibitory effect.

As used herein, the term “extract” refers to a substance obtained by separating from Gilgyeong ( Platycodon grandiflorum ).

In the present invention, the Gilgyeong ( Platycodon grandiflorum ) extract is extracted with a solvent selected from the group consisting of water, C1 to C4 alcohol, and a mixed solvent of water and C1 to C4 alcohol from leaves, roots, stems, etc. of Gilgyeong. It may be characterized as obtained, and preferably, the extract of Gilgyeong (Platycodon grandiflorum) of the present invention may be obtained by extracting water with a solvent.

The mixed solvent may preferably be about 70% ethanol aqueous solution. Extraction may be performed by an extraction method known in the art, for example, cold extraction, hot water extraction, ultrasonic extraction, reflux cooling extraction, etc., but is not limited thereto. The extraction temperature may be adopted by those skilled in the art in various temperature ranges suitable for the extraction method, for example, it may be carried out at 20 ℃ to 100 ℃, etc., but is not limited thereto. In addition, the extraction time is different depending on the extraction method, and a person skilled in the art may adopt an appropriate extraction time, but is not limited thereto, but may be performed single or multiple times in the range of about 1 hour to 10 days. Preferably, the extraction may be carried out by extracting with the above-mentioned extraction solvent 2 to 3 times at room temperature for about 2 days.

There is no limitation on the method for preparing the extract of the present invention, and any known method may be used. For example, the extract included in the composition of the present invention may be prepared in a powder state by an additional process such as distillation under reduced pressure and freeze-drying or spray-drying the primary extract extracted by the hot water extraction or solvent extraction method described above. In addition, the first extract was further purified using various chromatography methods such as silica gel column chromatography, thin layer chromatography, high performance liquid chromatography, etc. you may get Therefore, in the present invention, the extract is a concept including all extracts, fractions, and purified products obtained in each step of extraction, fractionation or purification, and their dilutions, concentrates, or dried products.

In the present invention, the gilgyeong extract may be characterized in that it is preferably in the form of granules, powders, or pellets.

In the present invention, the gilgyeong extract is preferably an extract extracted from the root and/or root bark of gilgyeong, but is not limited thereto, and may be extracted from leaves, stems, etc.

In the present invention, the composition may contain additional components necessary for administration, effect, etc., in addition to Gilkyung extract or Platicodin D, and may be used in combination with other coronavirus infection inhibitors, therapeutic agents, vaccines, neutralizing antibodies, etc. have.

Gilgyeong ( Platycodon grandiflorum ) extract of the present invention contains Platycodin D (Platycodin D) as an active ingredient.

The term “Platycodin D” of the present invention is one of the saponin components abundantly contained in the vicinity of the root of Gilkyung, and its specific chemical structure is as shown in Formula I below.

[Formula I]

As used herein, the term “salt” refers to an addition salt of an inorganic acid salt, an organic acid salt, or a metal salt of a compound, and may be a “pharmaceutically acceptable salt” when used in a pharmaceutical composition. The pharmaceutically acceptable salt may be a salt that does not cause serious irritation to the organism to which the compound is administered and does not impair the biological activity and properties of the compound. As used herein, the term “pharmaceutically acceptable salt” refers to a formulation of a compound that does not cause serious irritation to an organism to which the compound is administered and does not impair the biological activity and properties of the compound. The pharmaceutically acceptable salt is an acid that forms a non-toxic acid addition salt containing a pharmaceutically acceptable anion, for example, an inorganic acid such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrobromic acid, hydroiodic acid, etc., tartaric acid, formic acid , organic carbon acids such as citric acid, acetic acid, trichloroacetic acid, trifluoroacetic acid, gluconic acid, benzoic acid, lactic acid, fumaric acid, maleic acid, salicylic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, etc. Acid addition salts formed with the same sulfonic acid and the like are included. For example, pharmaceutically acceptable salts of carboxylic acids include metal salts or alkaline earth metal salts formed with lithium, sodium, potassium, calcium, magnesium, etc., amino acid salts such as lysine, arginine, and guanidine, dicyclohexylamine, N organic salts such as -methyl-D-glucamine, tris(hydroxymethyl)methylamine, diethanolamine, choline and triethylamine, and the like. Acids such as oxalic acid are not pharmaceutically acceptable, but may be used in the preparation of useful salts as intermediates for obtaining the compounds of the present invention and pharmaceutically acceptable salts thereof.

Gilgyeong extract of the present invention and its active ingredient, Platicodin D, are related to cell entry through the spike protein of coronavirus i) endocytosis by ACE2 and ii) direct fusion pathway by TMPRSS2 and ACE2 It may be characterized by suppression.

Gilgyeong extract and/or Platicodin D of the present invention may be characterized in that it inhibits or reduces the interaction of the TMPRSS2 and/or ACE2 with the coronavirus, in particular the spike protein of the coronavirus.

In one embodiment of the present invention, the coronavirus used SARS-CoV-2 virus, which is a virus that causes COVID-19, but a lenti recombined to express a spike protein similar to that of the SARS-CoV-2 virus. Inhibition of virus entry into cells was also confirmed. Thus, the Gilkyung extract and/or Platicodin D of the present invention expresses SARS-CoV-2 as well as a spike protein similar to SARS-CoV-2, which enters cells through interaction with ACE2 and/or TMPRSS2. It may be characterized in that it inhibits cell entry of other coronaviruses or viruses of other genera, and has the ability to prevent or treat the virus infection. For example, it is possible to effectively inhibit cell entry of MERS virus, SARS-CoV1 virus, and the like.

In the present invention, the "cell" refers to a cell capable of being infected with a virus, particularly a coronavirus. The cell may be a plant or animal cell, and may be an isolated cell or a cell not isolated from a living organism, organ, or tissue. Preferably, it may be characterized as a mammalian cell, more preferably a human cell. In the present invention, the cells are preferably respiratory cells, but are not limited thereto.

According to an embodiment of the present invention, the cells may include human lung epithelial cells (H1299, A549, Calu-3), for example, human lung fibroblasts (MRC-5), human colon (CaCo2), human It may include, but is not limited to, kidney (HEK293T), monkey kidney (Vero) cells, and the like, as long as it is a cell capable of obtaining the SARS-CoV-2 entry blocking effect by administration of the active ingredient or composition according to the present invention may be included in the above category.

In the pharmaceutical composition for preventing or treating severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection according to an embodiment of the present invention, the Platycodin D (Platycodin D) is the SARS-CoV-2 It may be to inhibit cell entry and/or inhibit binding to SARS-CoV-2 spike protein.

In the pharmaceutical composition for preventing or treating severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection according to an embodiment of the present invention, the Platycodin D (Platycodin D) is 0.001 based on the total weight of the composition to 50% by weight may be included.

A pharmaceutical composition for preventing or treating severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) according to the present invention includes an extract of Gilkyung ( Platycodon grandiflorum ).

The gilgyeong extract according to an embodiment of the present invention may further include oriental ingredients including deodeok extract, haengin extract, senega extract, licorice extract, etc., but is not limited thereto, and SARS-CoV-2 of the present invention Of course, if it is a component that can prevent or treat infection by blocking cell entry, it can be used by adding the enemy.

In the pharmaceutical composition for preventing or treating severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection according to an embodiment of the present invention, the Gilkyung ( Platycodon grandiflorum ) extract enters the cells of SARS-CoV-2 Inhibitory and/or inhibiting the binding to the SARS-CoV-2 spike protein may be.

In the pharmaceutical composition for preventing or treating severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection according to an embodiment of the present invention, the gilgyeong ( Platycodon grandiflorum ) extract is 0.001 to 50 based on the total weight of the composition Weight %, more preferably 1 to 30% by weight, more preferably 3 to 20% by weight may be included, but is not limited thereto, and if you can see the maximum preventive or therapeutic effect in a range that does not cause side effects, by adjusting appropriately Of course, it can be used.

The pharmaceutical composition may further include suitable carriers, excipients and diluents commonly used in pharmaceutical compositions in addition to containing the Platicodin D.

Carriers, excipients and diluents that may be included in the composition include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxy benzoate, propyl hydroxy benzoate, talc, magnesium stearate and mineral oil. When formulating the composition, it can be prepared using a diluent or excipient such as a filler, extender, binder, wetting agent, disintegrant, surfactant, etc. commonly used.

The pharmaceutical composition according to the present invention may be formulated and used in various forms according to conventional methods. Suitable dosage forms include oral dosage forms such as tablets, pills, powders, granules, dragees, hard or soft capsules, solutions, suspensions or emulsions, injections, and aerosols. This is most preferable. Examples of oral pharmaceuticals containing Gilgyeong extract include Yonggaksan. Other formulations include, but are not limited to, external preparations, suppositories, and sterile injection solutions.

The pharmaceutical composition according to the present invention can be prepared in a suitable dosage form using a pharmaceutically inert organic or inorganic carrier. That is, when the formulation is a tablet, a coated tablet, a dragee, and a hard capsule, it may contain lactose, sucrose, starch or a derivative thereof, talc, calcium carbonate, gelatin, stearic acid or a salt thereof. In addition, when the formulation is a soft capsule, it may contain vegetable oils, waxes, fats, semi-solid and liquid polyols. In addition, when the formulation is in the form of a solution or syrup, water, polyol, glycerol, and vegetable oil may be included.

The pharmaceutical composition according to the present invention may further include a preservative, a stabilizer, a wetting agent, an emulsifier, a solubilizing agent, a sweetener, a colorant, an osmotic pressure regulator, an antioxidant, and the like, in addition to the carrier described above.

The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount.

In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is the type, severity, and drug activity of the patient. , can be determined according to factors including sensitivity to drug, administration time, administration route and excretion rate, duration of treatment, concurrent drugs, and other factors well known in the medical field. The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or may be administered in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered single or multiple. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, which can be easily determined by those skilled in the art.

The composition of the present invention may be used in combination with various therapeutic agents and other pharmaceutical compositions for treating coronavirus infection that can be recognized by those skilled in the art as being effective in preventing or treating coronavirus infection.

The pharmaceutical composition of the present invention may be administered to an individual by various routes. The mode of administration may be, for example, oral administration. It may be administered by subcutaneous, intravenous, intramuscular or intrauterine intrathecal or intracerebrovascular injection. The pharmaceutical composition of the present invention is determined according to the type of drug as an active ingredient, along with several related factors such as the disease to be treated, the route of administration, the age, sex and weight of the patient, and the severity of the disease.

The present invention provides a pharmaceutical preparation comprising a pharmaceutical composition for preventing or treating the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection.

In the pharmaceutical formulation according to an embodiment of the present invention, the pharmaceutical formulation may be a solid dosage form or a liquid dosage form.

In the pharmaceutical preparation according to an embodiment of the present invention, the pharmaceutical preparation may be for oral, parenteral, nasal, oral, oral, sublingual, airway spray or rectal administration.

In the pharmaceutical formulation according to an embodiment of the present invention, the pharmaceutical formulation may be in the form of capsules, pills, tablets, solutions, suspensions, sprays, foams, patches or pastes.

The food composition for preventing or improving severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection according to the present invention contains Gilkyung ( Platycodon grandiflorum ) extract and/or Platycodin D (Platycodin D).

The gilgyeong extract according to an embodiment of the present invention may further include oriental ingredients including deodeok extract, haengin extract, senega extract, licorice extract, etc., but is not limited thereto, and SARS-CoV-2 of the present invention Of course, if it is a component that can prevent or treat infection by blocking cell entry, it can be used by adding the enemy.

In the food composition for preventing or improving severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection according to an embodiment of the present invention, the Gilgyeong ( Platycodon grandiflorum ) extract, Platycodin D (Platycodin D) Or the mixture may be included in an amount of 0.001 to 50% by weight, more preferably 1 to 30% by weight, and even more preferably 3 to 20% by weight, based on the total weight of the composition, but is not limited thereto, and is not limited thereto, as long as it does not cause side effects. Of course, if the preventive or therapeutic effect can be seen, it can be appropriately adjusted and used.

In another aspect, the present invention relates to a food for preventing or ameliorating a coronavirus infection comprising a gilgyeong extract and Platycodin D.

Food for the prevention or improvement of coronavirus infection inhibits the entry of coronavirus into cells or suppresses the cleavage of spike proteins important for infection in the viral packaging process, thereby inhibiting the infection of coronavirus and the proliferation of intracellular infectious islet coronavirus. It may be characterized as a health functional food having an activity to prevent infection of coronavirus or improve symptoms through the effect.

As used herein, the term “food” or “food composition” refers to meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, There are drinks, alcoholic beverages, vitamin complexes, health functional foods and health foods, and includes all foods in the ordinary sense.

The functional food (functional food) is the same term as food for special health use (FoSHU), and in addition to supplying nutrients, it is processed to efficiently exhibit bioregulatory functions and has high medical effects. means food. Here, "function (sex)" means to obtain a useful effect for health purposes such as regulating nutrients or physiological action with respect to the structure and function of the human body. The food of the present invention can be prepared by a method commonly used in the art, and at the time of manufacture, it can be prepared by adding raw materials and components commonly added in the art. In addition, the formulation of the food may be manufactured without limitation as long as it is a formulation recognized as a food, and the health functional food according to the present invention may be in the form of powder, granule, tablet, capsule or beverage.

The health food means food having an active health maintenance or promotion effect compared to general food, and health supplement food means food for the purpose of health supplementation. In some cases, the terms health functional food, health food, and dietary supplement are used interchangeably.

The food composition may further include a physiologically acceptable carrier, the type of carrier is not particularly limited and any carrier commonly used in the art may be used.

In addition, the composition may include additional ingredients that are commonly used in food compositions to improve odor, taste, vision, and the like. For example, vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, pantothenic acid, and the like may be included. Also, it may include minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu), and chromium (Cr). In addition, it may include amino acids such as lysine, tryptophan, cysteine, and valine.

In addition, the composition includes a preservative (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), a disinfectant (bleaching powder and high bleaching powder, sodium hypochlorite, etc.), an antioxidant (butylhydroxyanisole (BHA), butylhydroxy Toluene (BHT), etc.), coloring agents (tar pigments, etc.), coloring agents (sodium nitrite, sodium nitrite, etc.), bleach (sodium sulfite), seasonings (MSG, etc.), sweeteners (dulcin, cyclimate, saccharin, sodium, etc.) ), flavorings (vanillin, lactones, etc.), swelling agents (alum, D-potassium hydrogen tartrate, etc.), strengthening agents, emulsifiers, thickeners (foams), film agents, gum base agents, foam inhibitors, solvents, food additives such as improving agents additives). The additive may be selected according to the type of food and used in an appropriate amount.

It may further contain a food pharmaceutically acceptable food supplement additive together with any one or more of the Gilkyong extract and Platicodin D of the present invention, and may be used with other foods or food ingredients, and may be used appropriately according to a conventional method. can The mixed amount of the active ingredient may be suitably determined according to the purpose of its use (prevention, health or therapeutic treatment).

The method for preventing or treating severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection according to the present invention comprises the steps of administering an extract and / or Platycodin D ( Platycodin D) to a subject includes

In the method for preventing or treating severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection according to the present invention, the administration is oral, parenteral, nasal, oral, oral, sublingual, airway spray or rectal. it may be through

In the method for preventing or treating severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection according to the present invention, when the administration is performed by airway spray, the Platycodon grandiflorum extract and/or Platycodon Tycodin D (Platycodin D) may be applied to the respiratory tract to show the effect of preventing SARS-CoV-2 infection.

In the method for preventing or treating severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection according to the present invention, when performed by airway spray, the gilgyeong ( Platycodon grandiflorum ) extract, Platycodin D (Platycodin) D) or a mixture thereof may be included in an amount of 1 to 20% by weight based on the total weight of the composition.

In the method for preventing or treating severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection according to the present invention, the gilgyeong ( Platycodon grandiflorum ) extract may be in the form of granules or powder.

In the method for preventing or treating severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection according to the present invention, the gilgyeong ( Platycodon grandiflorum ) extract may be in the form of granules or powder for airway injection.

In the present invention, matters related to the mechanism of action of the gilgyeong extract and/or Platicodin D on SARS-CoV-2 may include all of the contents of published papers published after the original application of the present invention.

Hereinafter, the content of the present invention will be described in more detail through examples. The examples are only for explaining the present invention in more detail, and the scope of the present invention is not limited thereto.

[Virus, cell, plasmid and protocol]

- The Korean strain of SARS-CoV-2 (βCoV/KOR/KCDC03/2020) was provided by the Korea Centers for Disease Control and Prevention (KCDC).

- H1299, A549, MRC-5, and CaCo2 cells were obtained from the Korean Cell Line Bank.

- HEK293T cells were obtained from American Type Culture Collection (ATCC, USA, CRL-3216). For construction of SARS-CoV-2 spike-like lentivirus, HEK293T cells were infected with three plasmids. (Lentiviral backbone containing firefly luciferase and GFP expression cassette; psPAX2 packing plasmid containing Gag, Pol, Rev and Tat, SARS-CoV-2 spike plasmid).

- The pCEP4-myc-ACE2 plasmid was donated from Erik Procko (Addgene plasmid #141185).

- The pHR-CMV lentiviral expression vector is A. It was donated from A. Radu Aricescu (Addgene plasmid #113888).

- The TMPRSS2 lentiviral expression vector, RRL.sin.cPPT.SFFV/TMPRSS2 (variant 1).IRES-neo.WPRE (MT130) was donated by Caroline Goujon (Addgene plasmid # 145843).

[reagent]

-Platycodin D (PD), U18666A, E64d, chloroquine, camostat mesylate, nafamostat mesylate, ginsenoside Rb1, ginsenoside Rb3, Ginsenoside mix, glycyrrhizin, isoliquiritigenin, echinocystic acid, oleanolic acid, ursolic acid, and methyl-beta-cyclodextrin Sigma-Aldrich Co. (USA).

[protocol]

Protein extraction and immunoblot analysis

Cell lysates were prepared by solubilizing cells with ice-cold RIPA lysis buffer (Rockland Immunochemicals, USA) supplemented with a protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, USA) followed by centrifugation at 13,000 rpm for 20 min.

The clear cell lysate was mixed with an appropriate volume of 5x SDS sample buffer, separated by SDS-PAGE and transferred to a nitrocellulose membrane.

After blocking with 5% skim milk in TBST (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.05% Tween 20) for 1 h at room temperature (RT), the membrane was sealed with the following primary antibody at 4 °C. overnight in TBST in: rabbit anti-NPC1 (Novus, NB400-148), rabbit anti-NPC2 (Novus, NBP1-84012), rabbit anti-ACE2 (Abcam, ab15348), rabbit anti-TMPRSS2 (Abcam, ab92323) ), and mouse anti-GAPDH (Abcam, ab8245).

The corresponding horseradish peroxidase-conjugated secondary antibody (KPL, USA) was incubated for 1 h at room temperature.

Antibody-protein complexes were detected using ECL western blotting substrate (Thermo Fisher Scientific, USA).

Philippines III cholesterol staining

H1299 cells grown on coverslips were fixed with 4% PFA at room temperature for 10 min and then stained with 5 μg/ml filipin-III (Cayman, USA) in PBS/1% FBS solution for 2 h in the dark at room temperature. did. Stained cells were examined with an LSM700 confocal microscope (Carl Zeiss, Germany).

flow cytometry

Expi293F cells were harvested from pcDNA3-SARS-CoV-2-S-RBD-sfGFP (donated from Erik Procko, Addgene plasmid) using the ExpiFectamine™ 293 Transfection Kit (Thermo Fisher Scientific, USA) according to the manufacturer's instructions. # 141184) was used for transfection.

Cells were cultured for 4 to 5 days, then removed by centrifugation at 800 x g for 5 min, and the culture medium was stored at -4 °C.

To analyze the effect of PD on the binding of CoV-2-RBD-GFP to cell surface ACE2, H1299 cells expressing ACE2 were treated with DMSO or 5 μM PD for 1 h and washed with PBS-BSA on ice. .

Then incubated with 1/10 dilution of medium containing CoV-2-RBD-GFP for 30 min on ice. H1299 cells were then washed twice with PBS-BSA and analyzed with a BD LSRFortessa™ flow cytometer.

HPLC analysis

Gilgyeong ( Platycodon grandiflorum ) root or lysing acid (YGS) powder was dissolved in distilled water and HPLC system at 203 nm with RS Tech HECTOR-M C18 column (4.6 x 250 mm, 5 micron particle size, RS Tech Corp, Cheongju, South Korea) (Agilent 1100) was used for analysis.

The column was eluted at 25 °C with a mixture of solvent A, water, and solvent B, acetonitrile.

The concentration gradient elution system comprises 0 to 82:18/22 (A:B), 82:18/22 to 22:18 (A:B) to 70:30 (A:B), 32 at a flow rate of 1 mL/min. from 70:30 (A:B) to 50:50 (A:B) of minutes to 60 minutes.

Cell viability assay

H1299, Calu-3, Vero cells (5×10 ³ cells/well) seeded in 96-well plates were treated using the indicated concentrations of PD.

After 24 h treatment, WST-8 solution (Biomax, Korea) was added and incubated for 2 h.

The aqueous formazan formed in the culture medium was measured by a SpectraMax iD5 multi-mode microplate reader (Molecular Devices, USA) at 450 nm absorbance.

Relative cell viability (%) is expressed as a percentage relative to DMSO-treated control cells.

Molecular modeling of MβCD-containing complexes

MβCD binding complexes were prepared and scored using the Schrodinger Maestro software 2017 suite (Maestro, Schrodinger, LLC, New York, NY). Software tools were applied using default settings and pH 7.4 unless otherwise indicated below.

A binding complex model was prepared based on the crystal structure of the β-cyclodextrin (βCD) cholesterol-containing complex (CSD entry: KEXQUC). The crystal structure of tetradeca-2,6-O-methyl-βcyclodextrin (CSD entry: BOYFOK03) is used to represent MβCD in the binding complex. Both crystal structures were obtained from Cambridge Structural Database (CSD), and all water molecules were removed.

Ligands cholesterol and PD were represented by ChemDraw Professional 16.0 and introduced into the Maestro LigPrep module as structural data files. All ligands were prepared for further use using the Ligprep module: determination of ligand partial charge, optimization of geometry, and energy minimization using OPLS3 force fields were performed.

The MβCD binding complex was prepared using the βCD-cholesterol complex as a template.

First, the atomic coordinates of the two template βCD units were copied onto the two corresponding MβCD units. Cholesterol copied the atomic coordinates of the template explored cholesterol molecule and added it inside the complex, while PD was placed using a rigid alignment, maximum common structure molecular overlay tool.

To remove molecular strain and atomic bump and to account for the increase in macrocycle size as well as relaxation of the bonding complex structure, a constrained energy minimization algorithm was developed using the OPLS3 force field and 2 Å was applied using the heavy atom RMSD limit of

The obtained ligand binding poses were scored in situ by the Glide SP (standard precision) dock score, followed by prime MM using a variable-dielectric generalized Born (VDGB) continuous solvation model and an OPLS3 force field. - GBSA tool estimated relative ligand binding affinity.

Binding affinity was calculated using the following equation.

G _binding =E _complex (minimized)-(E _ligand (minimized) + E _{host (minimized)} ).

A higher negative score indicates a stronger coupling.

Binding complex visualization was performed using the Discovery Studio Client 2020 package (Dassault Systemes; BIOVIA. Discovery Studio Modeling Environment; Release 2020; Dassault Systemes: San Diego, CA, 2020).

PD and Cholesterol Orientation in Cell Membrane

1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphorus using the Discovery Studio Client 2020 package (Dassault Systemes; BIOVIA. Discovery Studio modeling environment; Release 2020; Dassault Systemes: San Diego, CA, 2020) The location and orientation of PD and cholesterol in the explicit membrane bilayer, consisting of poethanolamine (POPE) lipid molecules, was optimized.

Prior to developing membrane addition and molecular orientation algorithms, 3D conformations of PD and cholesterol were prepared using a standard ligand preparation protocol and using minimal energy using a generic CHARMm force field.

Molecules were first redirected to the implicit membrane by performing a stepwise search for the molecule's minimum solvation energy, calculated with the charmm36 force field and GBIM (Generalized Born Implicit Membrane) module. At this time, the film thickness was set to 35 angstroms.

After finding the optimal molecular position in the inward membrane, a lipid bilayer was constructed by adding POPE lipid molecules, water molecules and counterions (Na ⁺ and Cl ⁻ ) without performing system equilibration.

electrophysiology

For acute brain amputation, 5-week-old C57BL/6J mice were anesthetized with isoflurane and decapitated.

300 μm transverse slices of hippocampus were prepared in ice-cold NMDG-based cleavage solution (93 mM NMDG; 2.5 mM KCl; 1.2 mM NaH ₂ PO ₄ ; 30 mM NaHCO ₃ ; 20 mM HEPES; 25 mM glucose; 5 mM sodium ascorbate). , 2 mM tourea, 3 mM sodium pyruvate, 10 mM MgCl ₂ , 0.5 mM CaCl ₂ , adjusted to pH 7.3 with HCl, 310 mOsm) with DSK Linear Slicer Pro 7 (Dosaka EM Co., Ltd) prepared, and recovered after 15 minutes in the same solution at 32°C.

The obtained slices were recovered in oxygenated ACFS solution (126 mM NaCl; 24 mM NaHCO ₃ ; 2.5 mM KCl; 1 mM NaH ₂ PO ₄ ; 2 mM MgCl ₂ ; 10 mM glucose).

Spontaneous IPSCs were recorded under oxygenated ACFS solution with a whole-cell voltage clamp.

CsCl-based internal solutions (135 mM CsCl; 4 mM NaCl; 0.5 mM CaCl ₂ ; 10 to recording electrodes (6-8 MΩ) assembled from standard-wall borosilicate glass (GC150F-10, Warner Instrument Corp., USA). mM HEPES; 5 mM EGTA, 0.5 mM Na ₂ -GTP; 2 mM Mg-ATP; 1 mM QX-314).

For acute PD treatment and sIPSC recording, 10 μM PD-containing-ACSF was applied after establishing a baseline.

For long-term PD treatment and sIPSC recording, slices were incubated in PD-containing-ACSF (0, 0.3, 1, 3, 10, 30 μM, respectively) in the same solution for at least 1 h, and whole -cell voltage-clamp recorded.

sIPSCs were recorded at least 5 min. For sIPSC frequency and amplitude analysis, a small analysis program software (Synaptosoft) was used. Current maintenance below -300 pA in the baseline data was excluded for analysis.

statistical analysis

Data in the present invention are representative of two or three independent experiments with three samples and are presented as mean±SEM. Statistical analysis was performed using student t-test or one-way ANOVA followed by Tukey's post hoc test.

?, p-values less than 0.05 were considered statistically significant (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; NS, not significant not).

All statistical analysis was performed using Prism v.9.0.0 (GraphPad Software).

[Preparation Example 1] Cell culture

H1299 and A549 cells were cultured in RPMI-1640 (Gibco, USA) and MRC-5, CaCo2, and HEK293T cells were cultured in 10% fetal bovine serum (FBS; Gibco, USA) and 1X penicillin-streptomycin solution (HyClone, USA). Incubated in this supplemented DMEM medium (Dulbecco's Modified Eagle's Medium; Corning, USA) in a humidified 37 °C incubator containing 5% CO ₂ . For cell culture under lipoprotein-free conditions, lipoprotein-deficient FBS (Kalen biomedical, USA) was used.

For actual SARS-CoV-2 experiments, Vero cells were obtained from ATCC (CCL-81) and maintained at 37 °C with 5% CO in DMEM supplemented with FBS and 1X antibiotic-antifungal solution (Gibco, USA). .

Calu-3 used in the present invention is a clonal isolate, which shows a higher growth rate compared to the parental Calu-3 obtained from ATCC (HTB55). Calu-3 was maintained at 37 °C with 5% CO ₂ in EMEM medium (Eagle's Minimum Essential Medium; ATCC) supplemented with 10% FBS and 1X antibiotic-antifungal solution.

[Preparation Example 2] Establishment of plasmid and cell line

The full length of the ACE2 sequence from pCEP4-myc-ACE2 was cloned into the pHR-CMV lentiviral expression vector using Eco RI and Age 1 recognition sites.

For gene silencing, shRNA sequence targeting NPC1 (5'CCAGGTTCTTGACTTACAA-3'), NPC2 (5'-CGGTTCTGTGGATGGAGTTAT-3') and non-specific (NS) (5'-CAACAAGATGAAGAGCACCAA-3') containing Oligonucleotides were cloned into pLKO.1 puro lentiviral shRNA plasmid. For stable cell line generation, this plasmid was transiently transfected into HEK293T cells carrying the packaging plasmid psPAX2 and the envelope plasmid pMD2.G using Lipofectamine 3000 transfection reagent (Thermo Fisher, USA) according to the manufacturer's instructions.

At 24 and 48 hours post-transfection, the viral particle-containing supernatant was harvested, filtered through a 0.45 μm-pore-size filter and used to infect H1299 cells screened on 6-well plates and to 70-80% confluence. Incubated until reaching (confluence). 1 ml viral supernatant was directly overlaid on cells in the presence of polybrene (Merck, Germany) to a final concentration of 4 μg/ml. After 24 hours, the supernatant was replaced with a fresh medium and cultured for 2-3 days.

[Example 1] SARS-CoV-2 Spike-pseudotyped lentivirus production and transduction

SARS-CoV-2 spike-like lentivirus generation

SARS-CoV-2 spike (S)-like lentiviruses were generated using a second generation lentivirus packing system.

HEK293T cells that reached 70-80% confluency in 6-well plates were treated with Lipofectamine 3000 reagent (Invitrogen, USA) according to the manufacturer's instructions to express 1 µg of fluorescent protein and a firefly luciferase expression cassette, 0.75 μg of psPAX2 packing plasmid and 0.5 μg of SARS-CoV-2 spike plasmid (donated from Fang Li, Addgene plasmid #145032) were transfected.

The pMD2.G plasmid was used to generate a pseudotype control lentivirus with VSV-G. At 24 and 48 h post-transfection, the supernatant containing SARS-CoV-2 spike-like virus particles was harvested, filtered through a 0.45 um pore size filter, and stored at 4 °C until use. For long-term storage, it was stored at -80.

H1299 cells stably expressing ACE2 or ACE2 + TMPRSS2 were prepared as follows. H1299 cells were cultured in 48-well plates until 70-80% confluence was reached.

The viral supernatant was directly overlaid on H1299 cells in the presence of polybrene (Merck, Germany) at a final concentration of 4 μg/ml and cultured for at least 4 days.

For drug experiments, H1299 cells were pretreated with each drug for 24 hours before transduction. 48 hours after transfection, green fluorescnece protein (GFP) images were measured using an Olympus fluorescence microscope (Olympus), and transduction efficiency was measured using a luciferase assay system (Promega, USA) and a SpectraMax iD5 multi-mode microplate reader ( Molecular Devices, USA) was used to measure and quantify the activity of firefly luciferase in cell lysates (FIG. 1).

transduction assay

FIG. 2 shows the results of fluorescence microscopy and transduction efficiency measured by the above method.

As a result of observation under a fluorescence microscope, it was confirmed that a small number of H1299 cells were transduced with pseudovirus and exhibited GFP fluorescence. On the other hand, in H1299 cells stably expressing ACE2 (H1299-ACE2), the number of GFP-positive cells rapidly increased (Fig. 2a), and the SARS-CoV-2 spike protein was well integrated on the surface of the pseudovirus, and the actual virus It was confirmed that it can mimic cell entry of

In addition, as a result of measuring the transduction efficiency of the pseudovirus by measuring the luciferase activity, it was confirmed that the luciferase activity in H1299 and H1299-ACE cells increased 94 times and 4.4 x 10 ⁴ times, respectively, compared to the untransformed cells. and (Fig. 2b), from this, it was confirmed that H1299-ACE2 cells were more effective in SARS-CoV-2-S-induced host cell entry compared to H1299 parental cells.

[Example 2] Analysis of the inhibitory effect of platycodin D on SARS-CoV-2 entry into cells

To determine whether Platicodin D inhibits the entry of SARS-CoV-2 into cells, a SARS-CoV-2 spike-bearing pseudovirus was used.

First, H1299-ACE2 cells were transduced with SARS-CoV-2 spike-like lentiviral particles in the presence of DMSO or 10 μM Platicodin D (PD).

After culturing for 24 hours, the virus-containing supernatant was replaced with a fresh medium, and cultured for an additional 48 hours.

After incubation, fluorescence images were taken with a Nikon eclipse Ts2R fluorescence microscope, and by observing that GFP-positive cells decreased due to the treatment of PD, it was found that PD inhibited the entry of corona pseudovirus, and the results are shown in FIG. 3 .

In addition, after 1 hour treatment of H1299-ACE2 cells with DMSO or Platicodin D (PD) with 0.1, 0.3, 1, 3, 10 μM, vesicular stomatitis virus (VSV) glycoprotein in the presence of each concentration of PD -or transduced with SARS-CoV-2 spike-like lentiviral particles.

Transduction efficiency was quantified 24 hours after transduction through normalization to untreated control cells by measuring the activity of firefly luciferase in cell lysates.

The results are shown in FIG. 4 .

As a result of measuring the luciferase activity, it was confirmed that the SARS-CoV-2 cell entry was decreased depending on the amount of platicodin D treatment, and the IC ₅₀ was measured to be 0.69 μM.

In addition, it was confirmed that platicodin D does not block cell entry by the glycoprotein of vesicular stomatitis virus (VSV), and does not exhibit cytotoxicity to H1299 cells through a control experiment.

Comparison of the effect of saponin contained in ginseng to inhibit the entry into cells of SARS-CoV-2

For comparison with Platicodin D, a saponin component included in Gilgyeong, ginsenoside Rb1, Rb3, which are representative saponin components included in ginseng ( Panax ginseng ), and ginsenoside mix containing them in the experimental group For, as described above, SARS-Cov-2 pseudovirus transduction was performed to determine whether the inhibitory effect on the entry of SARS-CoV-2 into cells was measured.

The results are shown in FIG. 5 .

As can be seen from the results, it was confirmed that the saponin components contained in ginseng or a combination component thereof did not inhibit the entry into cells of SARS-CoV-2.

Identification of host cell entry inhibition pathway by platicodin D

In order to confirm the cell entry pathway of SARS-CoV-2 blocked by Platicodin D, H1299 cells expressing both ACE2 and TMPRSS2 (ACE2 / TMPRSS2 +), and cells expressing ACE2 alone (ACE2 +) were prepared, respectively. Then, the effect of platicodin D on these cells was measured.

Transduction efficiency was quantified 24 hours after transduction through normalization to untreated control cells by measuring the activity of firefly luciferase in cell lysates.

The results are shown in FIG. 6 .

As a result of luciferase analysis, E64d and chloroquine, inhibitors of lysosomal cathepsin, effectively blocked SARS-CoV-2 entry only in ACE2+ cells, but did not block entry in ACE/TMPRSS2+ cells. Conversely, TMPRSS2 inhibitors camostat and nafamostat effectively blocked SARS-CoV-2 entry only in ACE / TMPRSS2 + cells, but failed to block entry in ACE2+ cells Platicodin D was confirmed to effectively inhibit the entry of SARS-CoV-2 in both ACE2+ cells and ACE/TMPRSS2+ cells. (Fig. 6).

This result was confirmed to appear in a similar form in ACE2+ and ACE2 / TMPRSS2 + cells for HEK239T cells instead of H1299.

In H1299 cells expressing both ACE2 and TMPRSS2 (ACE2 / TMPRSS2 +), the IC ₅₀ of platicodin D was measured to be 0.72 μM, respectively.

[Example 3] Inhibitory effect of SARS-CoV-2 in cell entry of Yonggaksan

Yonggaksan (YGS) powder (Ingredients: Gilgyeong extract 11.7mg, Polygala senega root extract 0.5mg, apricot kernel oil 0.83mg, and licorice powder 8.3mg) is dissolved in 10 ml DMEM medium and heated at room temperature for 30 minutes. 15 mg/ml Yonggaksan stock solution was prepared by placing it on a shaker. After centrifugation at 2,000 rpm for 5 minutes, the supernatant was filtered through a syringe filter with a 0.45 mm-pore size to further remove particulate matter from the solution.

ACE2+ and ACE / TMPRSS2 + H1299 cells were transduced with SARS-CoV-2 spike-like virus after 1 hour of treatment by sequentially diluting 15 mg/ml ryukaksan solution, and ryukaksan was added at the same concentration during transduction. After 24 hours, the activity of firefly luciferase was measured.

The result is shown in FIG. 7A.

The lysate dilution solution effectively inhibited the entry of SARS-CoV-2 into ACE2+ and ACE/TMPRSS2+ cells in a dose-dependent manner, and the IC50 for each case was 3.17 and 3.72 mg/ml.

Effect of treatment after serial dilution of Ryukakusan

The recommended daily amount of ryukaksan preparation for adults is 0.3g 3-6 times a day. For imitation, before SARS-CoV-2 pseudovirus was added, ryukaksan was diluted 4 times for 20 minutes at 4 hour intervals in H1299-ACE2 cells. was processed. After transfection, no ryukaksan was added.

Transduction efficiency was quantified 24 hours after transduction through normalization to untreated control cells by measuring the activity of firefly luciferase in cell lysates.

It was confirmed that SARS-CoV-2 infection was effectively blocked even when the pretreatment was repeated for such a short time, and at this time, the IC50 for ACE2+ and ACE/TMPRSS2+ cells was 13.49 and 15.26 mg/ml . was measured as The results are shown in Fig. 7b.

From the above results, it was confirmed that Yonggaksan was very effective in blocking SARS-CoV-2 entry.

Measurement of the inhibition of SARS-CoV-2 entry into cells by substances other than Gilkyong extracts contained in Yonggaksan

Glycyrrhizin, which is known as the active substance of licorice powder, which is the main ingredient other than gilgyeong powder, in Yonggaksan and

It was confirmed whether isoricuritigenin inhibited the entry into cells of SARS-CoV-2, but it was not effective at all and it was confirmed that there was no effect of enhancing the anti-coronavirus activity of Platicodin D.

The results are shown in Fig. 7c.

[Example 4] Inhibitory effect on intracellular entry of SARS-CoV-2 of bellflower branch

ACE2+ and ACE / TMPRSS2 + H1299 cells were transduced with SARS-CoV-2 spike-like virus after 1 hour treatment by sequentially diluting a solution of bellflower root containing bellflower root at a concentration of 15 mg/ml. At the same concentration, a solution of bellflower branch was added. After 24 hours, the activity of firefly luciferase was measured.

The result is shown in FIG.

The diluent ryukaksan effectively inhibited the entry of SARS-CoV-2 into ACE2+ and ACE/TMPRSS2+ cells in a dose-dependent manner, and the IC50 for each case was 2.91 and 3.69 mg/ml.

From the above results, it was confirmed that foods containing bellflower root, such as bellflower green, were very effective in blocking SARS-CoV-2 entry.

[Example 5] Determination of whether Platicodin D is a major component in inhibiting the flow and entry of SARS-CoV-2 into cells in Yonggaksan and Bellflower roots

High-performance liquid chromatography (HPLC) analysis was performed using 750 mg of ryukaksan, 660 mg of bellflower root, and 0.25 mg of platycodin D to confirm the peak of platicodin D and obtain the peak area value. (Fig. 9a).

A quantification curve of Platicodin D was drawn through an external standard method to obtain the formula value “Y=2356*X-220.1”, and the coefficient of determination (R2) was 0.9989 (FIG. 9b).

Platicodin D content in Yonggaksan and bellflower root was obtained by substituting the area value of Platicodin D obtained from HPLC into this formula value, and the results were calculated as 0.02% and 0.018%, respectively (FIG. 9a).

A SARS-CoV-2 entry assay was performed on H1299 (ACE2+) cells with the same ryukaksan and bellflower root extract solution, and the "converted IC50" was calculated by substituting the content of Platicodin D obtained above.

The converted IC50s were calculated to be 0.99 μM and 0.7 μM in the extracts of ryukaksan and bellflower root, which were almost identical to the IC50 of 0.69 μM obtained from the single substance of Platicodin D.

Through this, it was confirmed that the main substance that inhibits the entry of coronavirus in Yonggaksan and bellflower root is Platicodin D.

[Example 6] ACE2 binding inhibitory ability analysis of platicodin D to SARS-CoV-2 spike protein receptor binding domain (RBD)

To determine whether platicodin D inhibits the binding of ACE2 to the SARS-CoV-2 spike protein receptor binding domain (RBD), an experiment was performed with the following procedure.

First, H1299-ACE2 cells were cultured in a medium containing the receptor binding domain of SARS-CoV-2 fused with GFP.

The plasmid for the production of CoV-2-RBD-GFP was transfected into Expi293F cells using Expifectamine according to the manufacturer's instructions. A transfection enhancer was added 18 hours after transduction, and the cells were cultured for 4-5 days. Cells were removed by centrifugation at 800 x g for 5 min, and the medium was stored at -4 °C.

To analyze the binding of CoV-RBD-GFP to cell surface ACE2, H1299-myc-ACE2 cells were washed with cold PBS-BSA, diluted 1/10 with a solution containing CoV-2-RBD-GFP and 1/ A dilution of anti-myc Alexa 647 diluted with 240 was added and incubated on ice for 30 minutes. Cells were washed twice with PBS-BSA and analyzed with a BD LSRFortessa ^™ flow cytometer.

The results are shown in FIG. 10 .

Through flow cytometry analysis, it was confirmed that 95% of SARS-CoV-2-RBD-GFP could bind to H1299-ACE2 cells. It was confirmed that -RBD-GFP binding was significantly reduced.

In addition, from the double-color flow cytometry results, it was confirmed that ACE2 was reduced on the cell surface through platicodin D (PD) treatment, which could reduce the binding of SARS-CoV-RBD-GFP to H1299-ACE2 cells. did

[Example 7] Confirmation of spike protein cleavage inhibitory effect during SARS-CoV-2 virus packaging of Platicodin D

In the viral packaging process, furin protease cleaves the SARS-CoV-2 spike protein at the S1/S2 subunit boundary, and this cleavage process plays an important role in SARS-CoV-2 infectivity. in charge

In order to analyze the effect of Platicodin D on the purine protease, when cells produce SARS-CoV-2 pseudovirus, HEK293T cells are treated with Platicodin D, and then virus entry is analyzed and the results are shown. 11 is shown.

5 μM PD was added to HEK293T cells 6 hours after transfection of the viral vector. The supernatant containing SARS-Cov-2 pseudovirus was collected after additional incubation for 48 hours. Viruses obtained from PD-treated HEK293T cells were subjected to a pseudovirus entry assay. PD-treated HEK293T cells were analyzed by western blot to detect cleavage of the SARS-CoV-2 spike (red arrow) using an anti-C9 antibody targeting the C-terminal C9 tag of the spike protein. As a control group, an experimental group treated only with DMSO was used.

From the above results, it was confirmed that Platicodin D significantly reduced the infection of the virus. In addition, from the western blot test, it was confirmed that platicodin D inhibited the cleavage of SARS-CoV-2 protein in virus packaging HEK293T cells.

[Example 8] Analysis of real SARS-CoV-2 virus and dose-response curves by immunofluorescence assay

A Korean strain of SARS-CoV-2 (βCoV/KOR/KCDC03/2020) was provided by KCDC and used, and the virus titer was determined by plaque assay in Vero (TMPRSS2-) cells. All experiments with SARS-CoV-2 were conducted in accordance with the guidelines of KNH, using the Enhanced Biosafety Level 3 (BSL-3) restriction procedure in a laboratory approved for use by the KCDC, and the Institute of Pasteur, Korea (Institut Pasteur). Korea).

24 h before the experiment, Vero (TMPRSS2-) cells were seeded at 1.2 × 10 ⁴ cells per well in assay 384-well, μClear plate (Greiner Bio-One, Austria), and Calu-3 (TMPRSS2+) cells were 2.0 × 10 ⁴ cells were seeded per well.

A 10-point dose-dependent curve (DRC) was generated using compound concentrations ranging from 0.02-10 μM and 0.05-24 mg/ml. However, chloroquine and remdesivir were used in the range of 0.1-150 μM.

For viral infection, plates were transferred to a BSL-3 restriction facility and SARS-CoV-2 was added at a multiplicity of infection (MOI) of 0.0125 in Vero cells and 0.5 in Calu-3 cells.

Cells were fixed at 24 hpi using 4% paraformaldehyde (PFA), stained with SARS-CoV-2 nucleocapsid protein (N protein), and analyzed by immunofluorescence assay.

Acquired images were analyzed with Columbus software (PerkinElmer, Inc. Waltham, MA) to quantify cell number and infection rate, and antiviral activity was positive (mimetic) and negative (0.5%) in each assay plate. DMSO) normalized to control.

DRC was established as a regression model using XLfit 4 software or Prism 6 by a sigmoidal dose-response model with the following equation.

Y = lower + (upper lower)/(1 + (IC ₅₀ /X)hillslope).

Maximum half inhibitory concentration (IC ₅₀ ) values were calculated from normalized activity dataset-regression. IC ₅₀ was measured in duplicate and the quality of each assay was controlled by the Z'-factor and percent coefficient of change (%CV).

At this time, chloroquine, lopinavir, and remdesivir were utilized as reference drugs having IC ₅₀ values of 7.91, 22.65, and 10.67 μM, respectively. It was confirmed that chloroquine was effective only on Vero cells containing TMPRSS2-, and the TMPRSS2 inhibitors Chamostat and Napamostat showed strong antiviral effects on TMPRSS2+ Calu-3 cells, but had little effect on Vero cells.

On the other hand, platicodin D according to the present invention showed an antiviral effect against SARS-CoV-2 with IC ₅₀ values of 1.19 μM and 4.76 μM, respectively, in both Vero and Calu-3 cells, so that platicodin D was found in lysosome and It was newly confirmed that there was an effect of effectively blocking the SARS-CoV-2 entry pathway driven by TMPRSS2 ( FIG. 12 ).

The IC ₅₀ measurement results for the experimental drugs are shown in Table 1 below.

Drug IC ₅₀ in Vero (μM) IC ₅₀ in Calu3 (μM) Platycodin D 1.19 4.76 Chloroquine 7.91 > 50 ^a Camostat > 50 ^a 0.187 ^a Nafamostat 13.88 ^a 0.0022 ^a Remdesivir 10.67 1.74 Lopinavir 22.65 21.7 ^{a a} Ko M et al. J Med Virol. 2020 Aug 7:10.1002/jmv.26397.

[Example 9] Confirmation of inhibition of SARS-CoV-2 entry through changes in intracellular cholesterol of Platicodin D

In H1299 (ACE2+) cells, it was confirmed by filipin staining that the location and amount of cholesterol in the cells were changed during PD treatment, and the results are shown in FIG. 13A .

Changes in intracellular cholesterol can inhibit the entry of SARS-CoV-2, which means that the NPC1 protein located in the lysosome, which plays an important role in intracellular cholesterol distribution, is lost through shRNA or the function can be inhibited by treating the NPC1 inhibitor U18666A. In this case, it was confirmed by observing that the infectivity of SARS-CoV-2 was lowered, and the results are shown in FIG. 13B.

In addition, it was observed that the SARS-CoV-2 entry inhibitory ability of PD in H1299 (ACE2+) cells was completely inhibited by Mβ (methylbeta cyclodextrin), a cholesterol chelate ( FIG. 13a ).

It was confirmed through molecular modeling of the Mβ-containing complex that Mβ can chelate not only cholesterol but also platicodin D, and the Glide G score showed that Mβ had a higher binding affinity for platicodin D than cholesterol. The state in which Platicodin D is chelated to Mβ is schematically shown in FIG. 14 .

[Example 10] Platicodin D cell membrane orientation molecular modeling

Through molecular modeling, it was confirmed that platicodin D can be placed inside the cell membrane, and the orientation of platicodin D and cholesterol in the cell membrane is schematically shown in FIG. 15 .

[Example 11] Platicodin D membrane fusion inhibitory activity

The effect of PD on inhibitory post-synaptic currents (sIPSCs) in brain slices, a well-known membrane fusion phenomenon, was tested. Slice patch clamp recordings were performed on hippocampal CA1 pyramidal neurons and sIPSCs were measured before and after PD treatment. As a result, it was confirmed that PD effectively suppressed the frequency of sIPSC without affecting the amplitude, and the IC ₅₀ of PD at this time was 6.724 μM.

The results are shown in FIG. 16 .

This supports the fact that the inhibitory action of PD on pSARS-CoV-2-entry is due to its ability to prevent membrane fusion.

[Example 12] Confirmation of SARS-CoV-2 virus entry blocking effect through autophagy control of platicodin D

As a result of treating A549 cells and H1299 cells with rapamycin, an autophagy inducer, it was confirmed that there was no blocking effect on SARS-CoV-2 entry in H1299 cells, and some blocking effect on A549 cells.

In addition, when the expression of ATG5, which is very important for autophagosome formation, was lost in H1299 cells through shRNA, there was no effect on the inhibitory effect of SARS-CoV-2 on the cell entry of PD. It was confirmed that the cell entry inhibitory effect of SARS-CoV-2 was almost completely offset.

The results are shown in FIG. 17 .

The above results show that autophagy is involved in the inhibition of SARS-CoV-2 entry by PD, and this phenomenon was observed only in A549 cells, so it can be inferred that the details of the infection mechanism may differ from cell to cell.

From the above results, the viral entry and infection inhibition mechanisms of Platicodin D are largely i) inhibition of the interaction of ACE2 with SARS-CoV-2, ii) inhibition of the cellular entry process of SARS-CoV-2, and iii) SARS- In the intracellular virus packaging process of CoV-2, it is divided into inhibition of the production of infectious SARS-CoV-2 through inhibition of spike protein cleavage, ii) inhibition of the entry process of SARS-CoV-2 into the cell, Again (a) cleavage of the spike protein by TMPRSS2 and (b) after endocytosis, cathepsin B/L-mediated cleavage of the spike protein in the lysosome shows the inhibitory effect of virus-cell membrane fusion confirmed that.

As described above in detail a specific part of the content of the present invention, for those of ordinary skill in the art, this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby It will be obvious. Accordingly, it is intended that the substantial scope of the present invention be defined by the appended claims and their equivalents.

<110> Institute for Basic Science <120> Pharmaceutical composition for the prevention and treatment of Envelopled viruses comprising taining Platycodon grandiflorus root extracts <130> P20091651130 <150> KR 10-2020-0099311 <151> 2020-08-07 <160> 3 <170> KoPatentIn 3.0 <210> 1 <211> 12289 <212> DNA <213> Artificial Sequence <220> <223> Lentiviral backbone that contains expression cassettes for firefly luciferase and GFP <400> 1 ctagaattaa ttccgtgtat tctatagtgt cacctaaatc gtatgtgtat gatacataag 60 gttatgtatt aattgtagcc gcgttctaac gacaatatgt acaagcctaa ttgtgtagca 120 tctggcttac tgaagcagac cctatcatct ctctcgtaaa ctgccgtcag agtcggtttg 180 gttggacgaa ccttctgagt ttctggtaac gccgtcccgc acccggaaat ggtcagcgaa 240 ccaatcagca gggtcatcgc tagccagatc ctctacgccg gacgcatcgt ggccggcatc 300 accggcgcca caggtgcggt tgctggcgcc tatatcgccg acatcaccga tggggaagat 360 cgggctcgcc acttcgggct catgagcgct tgtttcggcg tgggtatggt ggcaggcccc 420 gtggccgggg gactgttggg cgccatctcc ttgcatgcac cattccttgc ggcggcggtg 480 ctcaacggcc tcaacctact actgggctgc ttcctaatgc aggagtcgca taagggagag 540 cgtcgaatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg 600 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta 660 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc 720 gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat 780 aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat 840 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata 900 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct 960 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa 1020 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa 1080 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt 1140 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg 1200 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca 1260 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa 1320 cactgcggcc aacttacttc tg acaacgat cggaggaccg aaggagctaa ccgctttttt 1380 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc 1440 cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa 1500 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga 1560 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc 1620 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga 1680 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga 1740 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga 1800 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat 1860 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt 1920 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct 1980 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc 2040 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc 2100 aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc 2160 gcctacatac ctcgctctgc taatcctg tt accagtggct gctgccagtg gcgataagtc 2220 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg 2280 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata 2340 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta 2400 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc 2460 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg 2520 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt 2580 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt 2640 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga 2700 gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc 2760 cgcgcgttgg ccgattcatt aatgcagctg tggaatgtgt gtcagttagg gtgtggaaag 2820 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc 2880 aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat 2940 tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt 3000 tccgcccatt ctccgcccca tggctgacta att tttttta tttatgcaga ggccgaggcc 3060 gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt 3120 tgcaaaaagc ttggacacaa gacaggcttg cgagatatgt ttgagaatac cactttatcc 3180 cgcgtcaggg agaggcagtg cgtaaaaaga cgcggactca tgtgaaatac tggtttttag 3240 tgcgccagat ctctataatc tcgcgcaacc tattttcccc tcgaacactt tttaagccgt 3300 agataaacag gctgggacac ttcacatgag cgaaaaatac atcgtcacct gggacatgtt 3360 gcagatccat gcacgtaaac tcgcaagccg actgatgcct tctgaacaat ggaaaggcat 3420 tattgccgta agccgtggcg gtctgtaccg ggtgcgttac tggcgcgtga actgggtatt 3480 cgtcatgtcg ataccgtttg tatttccagc tacgatcacg acaaccagcg cgagcttaaa 3540 gtgctgaaac gcgcagaagg cgatggcgaa ggcttcatcg ttattgatga cctggtggat 3600 tccggtggta ctgcggttgc gattcgtgaa atgtatccaa aagcgcactt tgtcaccatc 3660 ttcgcaaaac cggctggtcg tccgctggtt gatgactatg ttgttgatat cccgcaagat 3720 acctggattg aacagccgtg ggatatgggc gtcgtattcg tcccgccaat ctccggtcgc 3780 taatcttttc aacgcctggc actgccgggc gttgttcttt ttaacttcag gcgggttaca 3840 atagtttcca gtaagtattc tggaggctgc atccatgac a caggcaaacc tgagcgaaac 3900 cctgttcaaa ccccgcttta aacatcctga aacctcgacg ctagtccgcc gctttaatca 3960 cggcgcacaa ccgcctgtgc agtcggccct tgatggtaaa accatccctc actggtatcg 4020 catgattaac cgtctgatgt ggatctggcg cggcattgac ccacgcgaaa tcctcgacgt 4080 ccaggcacgt attgtgatga gcgatgccga acgtaccgac gatgatttat acgatacggt 4140 gattggctac cgtggcggca actggattta tgagtgggcc ccggatcttt gtgaaggaac 4200 cttacttctg tggtgtgaca taattggaca aactacctac agagatttaa agctctaagg 4260 taaatataaa atttttaagt gtataatgtg ttaaactact gattctaatt gtttgtgtat 4320 tttagattcc aacctatgga actgatgaat gggagcagtg gtggaatgcc tttaatgagg 4380 aaaacctgtt ttgctcagaa gaaatgccat ctagtgatga tgaggctact gctgactctc 4440 aacattctac tcctccaaaa aagaagagaa aggtagaaga ccccaaggac tttccttcag 4500 aattgctaag ttttttgagt catgctgtgt ttagtaatag aactcttgct tgctttgcta 4560 tttacaccac aaaggaaaaa gctgcactgc tatacaagaa aattatggaa aaatattctg 4620 taacctttat aagtaggcat aacagttata atcataacat actgtttttt cttactccac 4680 acaggcatag agtgtctgct attaataact atgctcaaaa attg tgtacc tttagctttt 4740 taatttgtaa aggggttaat aaggaatatt tgatgtatag tgccttgact agagatcata 4800 atcagccata ccacatttgt agaggtttta cttgctttaa aaaacctccc acacctcccc 4860 ctgaacctga aacataaaat gaatgcaatt gttgttgtta acttgtttat tgcagcttat 4920 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg 4980 cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg gatcaactgg 5040 ataactcaag ctaaccaaaa tcatcccaaa cttcccaccc cataccctat taccactgcc 5100 aattacctgt ggtttcattt actctaaacc tgtgattcct ctgaattatt ttcattttaa 5160 agaaattgta tttgttaaat atgtactaca aacttagtag ttggaagggc taattcactc 5220 ccaaagaaga caagatatcc ttgatctgtg gatctaccac acacaaggct acttccctga 5280 ttagcagaac tacacaccag ggccaggggt cagatatcca ctgacctttg gatggtgcta 5340 caagctagta ccagttgagc cagataaggt agaagaggcc aataaaggag agaacaccag 5400 cttgttacac cctgtgagcc tgcatgggat ggatgacccg gagagagaag tgttagagtg 5460 gaggtttgac agccgcctag catttcatca cgtggcccga gagctgcatc cggagtactt 5520 caagaactgc tgatatcgag cttgctacaa gggactttcc gctggggact ttccagggag 5580 gcgtggcctg ggcgggactg gggagtggcg agccctcaga tcctgcatat aagcagctgc 5640 tttttgcctg tactgggtct ctctggttag accagatctg agcctgggag ctctctggct 5700 aactagggaa cccactgctt aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt 5760 gtgcccgtct gttgtgtgac tctggtaact agagatccct cagacccttt tagtcagtgt 5820 ggaaaatctc tagcagtggc gcccgaacag ggacttgaaa gcgaaaggga aaccagagga 5880 gctctctcga cgcaggactc ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg 5940 actggtgagt acgccaaaaa ttttgactag cggaggctag aaggagagag atgggtgcga 6000 gagcgtcagt attaagcggg ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc 6060 agggggaaag aaaaaatata aattaaaaca tatagtatgg gcaagcaggg agctagaacg 6120 attcgcagtt aatcctggcc tgttagaaac atcagaaggc tgtagacaaa tactgggaca 6180 gctacaacca tcccttcaga caggatcaga agaacttaga tcattatata atacagtagc 6240 aaccctctat tgtgtgcatc aaaggataga gataaaagac accaaggaag ctttagacaa 6300 gatagaggaa gagcaaaaca aaagtaagac caccgcacag caagcggccg gccgctgatc 6360 ttcagacctg gaggaggaga tatgagggac aattggagaa gtgaattata taaat ataaa 6420 gtagtaaaaa ttgaaccatt aggagtagca cccaccaagg caaagagaag agtggtgcag 6480 agagaaaaaa gagcagtggg aataggagct ttgttccttg ggttcttggg agcagcagga 6540 agcactatgg gcgcagcgtc aatgacgctg acggtacagg ccagacaatt attgtctggt 6600 atagtgcagc agcagaacaa tttgctgagg gctattgagg cgcaacagca tctgttgcaa 6660 ctcacagtct ggggcatcaa gcagctccag gcaagaatcc tggctgtgga aagataccta 6720 aaggatcaac agctcctggg gatttggggt tgctctggaa aactcatttg caccactgct 6780 gtgccttgga atgctagttg gagtaataaa tctctggaac agatttggaa tcacacgacc 6840 tggatggagt gggacagaga aattaacaat tacacaagct taatacactc cttaattgaa 6900 gaatcgcaaa accagcaaga aaagaatgaa caagaattat tggaattaga taaatgggca 6960 agtttgtgga attggtttaa cataacaaat tggctgtggt atataaaatt attcataatg 7020 atagtaggag gcttggtagg tttaagaata gtttttgctg tactttctat agtgaataga 7080 gttaggcagg gatattcacc attatcgttt cagacccacc tcccaacccc gaggggaccc 7140 gacaggcccg aaggaataga agaagaaggt ggagagagag acagagacag atccattcga 7200 ttagtgaacg gatctcgacg gtatcgccaa atggcagtat tcatccacaa ttttaaaaga 7260 aaagggggga ttggggggta cagtgcaggg gaaagaatag tagacataat agcaacagac 7320 atacaaacta aagaattaca aaaacaaatt acaaaaattc aaaattttcg ggtttattac 7380 agggacagca gagatccagt ttggatcgat aagcttgata tcgaattgct gcagccccga 7440 tagtcgacta catttatatt ggctcatgtc caatatgacc gccatgttga cattgattat 7500 tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt 7560 tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc 7620 cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac 7680 gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata 7740 tgccaagtcc gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc 7800 agtacatgac cttacgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta 7860 ttaccatggt gatgcggttt tggcagtaca ccaatgggcg tggatagcgg tttgactcac 7920 ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc 7980 aacgggactt tccaaaatgt cgtaataacc ccgccccgtt gacgcaaatg ggcggtaggc 8040 gtgtacggtg ggaggtctat ataagcagag ctctccctat cagtgataga gatctcccta 8100 t cagtgatag agatcgtcgt cgtgctcgtt tagtgaaccg tctgatctca acaagctgtc 8160 tagagaattc gccaccatgg aagacgccaa aaacataaag aaaggcccgg cgccattcta 8220 tccgctggaa gatggaaccg ctggagagca actgcataag gctatgaaga gatacgccct 8280 ggttcctgga acaattgctt ttacagatgc acatatcgag gtggacatca cttacgctga 8340 gtacttcgaa atgtccgttc ggttggcaga agctatgaaa cgatatgggc tgaatacaaa 8400 tcacagaatc gtcgtatgca gtgaaaactc tcttcaattc tttatgccgg tgttgggcgc 8460 gttatttatc ggagttgcag ttgcgcccgc gaacgacatt tataatgaac gtgaattgct 8520 caacagtatg ggcatttcgc agcctaccgt ggtgttcgtt tccaaaaagg ggttgcaaaa 8580 aattttgaac gtgcaaaaaa agctcccaat catccaaaaa attattatca tggattctaa 8640 aacggattac cagggatttc agtcgatgta cacgttcgtc acatctcatc tacctcccgg 8700 ttttaatgaa tacgattttg tgccagagtc cttcgatagg gacaagacaa ttgcactgat 8760 catgaactcc tctggatcta ctggtctgcc taaaggtgtc gctctgcctc atagaactgc 8820 ctgcgtgaga ttctcgcatg ccagagatcc tatttttggc aatcaaatca ttccggatac 8880 tgcgatttta agtgttgttc cattccatca cggttttgga atgtttacta cactcggata 8940 tttgata tgt ggatttcgag tcgtcttaat gtatagattt gaagaagagc tgtttctgag 9000 gagccttcag gattacaaga ttcaaagtgc gctgctggtg ccaaccctat tctccttctt 9060 cgccaaaagc actctgattg acaaatacga tttatctaat ttacacgaaa ttgcttctgg 9120 tggcgctccc ctctctaagg aagtcgggga agcggttgcc aagaggttcc atctgccagg 9180 tatcaggcaa ggatatgggc tcactgagac tacatcagct attctgatta cacccgaggg 9240 ggatgataaa ccgggcgcgg tcggtaaagt tgttccattt tttgaagcga aggttgtgga 9300 tctggatacc gggaaaacgc tgggcgttaa tcaaagaggc gaactgtgtg tgagaggtcc 9360 tatgattatg tccggttatg taaacaatcc ggaagcgacc aacgccttga ttgacaagga 9420 tggatggcta cattctggag acatagctta ctgggacgaa gacgaacact tcttcatcgt 9480 tgaccgcctg aagtctctga ttaagtacaa aggctatcag gtggctcccg ctgaattgga 9540 atccatcttg ctccaacacc ccaacatctt cgacgcaggt gtcgcaggtc ttcccgacga 9600 tgacgccggt gaacttcccg ccgccgttgt tgttttggag cacggaaaga cgatgacgga 9660 aaaagagatc gtggattacg tcgccagtca agtaacaacc gcgaaaaagt tgcgcggagg 9720 agttgtgttt gtggacgaag taccgaaagg tcttaccgga aaactcgacg caagaaaaat 9780 cagagagatc ct cataaagg ccaagaaggg cggaaagatc gccgtgtaaa ccggtgcatc 9840 tggaagtggt accctggagg tgctgttcca gggccccgga ggttccggtg gttccggtct 9900 gaatgatatc tttgaagctc agaagattga atggcatgaa ggacgtacca agcaccacca 9960 tcaccatcac taatgatcac tcgagcgccc ctctccctcc ccccccccta acgttactgg 10020 ccgaagccgc ttggaataag gccggtgtgc gtttgtctat atgttatttt ccaccatatt 10080 gccgtctttt ggcaatgtga gggcccggaa acctggccct gtcttcttga cgagcattcc 10140 taggggtctt tcccctctcg ccaaaggaat gcaaggtctg ttgaatgtcg tgaaggaagc 10200 agttcctctg gaagcttctt gaagacaaac aacgtctgta gcgacccttt gcaggcagcg 10260 gaacccccca cctggcgaca ggtgcctctg cggccaaaag ccacgtgtat aagatacacc 10320 tgcaaaggcg gcacaacccc agtgccacgt tgtgagttgg atagttgtgg aaagagtcaa 10380 atggctctcc tcaagcgtat tcaacaaggg gctgaaggat gcccagaagg taacccattg 10440 tatgggatct gatctggggc ctcggtacac atgctttaca tgtgtttagt cgaggttaaa 10500 aaaacgtcta ggccccccga accacgggga cgtggttttc ctttgaaaaa cacgatgata 10560 atatggccac aaccatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc 10620 tggtcgag ct ggacggcgac gtaaacggcc acaagttcag ctgtccggcg agggcgaggg 10680 cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt 10740 gccctggccc accctcgtga ccaccttgac ctacggcgtg cagtgcttcg cccgctaccc 10800 cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga 10860 gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga 10920 gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa 10980 catcctgggg cacaagctgg agtacaacta caacagccac aaggtctata tcaccgccga 11040 caagcagaag aacggcatca aggtgaactt caagacccgc cacaacatcg aggacggcag 11100 cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct 11160 gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg 11220 cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga 11280 gctgtacaag taaagcggcc gcgactcttg agtcgagctg caggcatgca agcttgatat 11340 cgtacgtatc gataatcaac ctctggatta caaaatttgt gaaagattga ctggtattct 11400 taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc 11460 tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct 11520 ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga 11580 cgcaaccccc actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc 11640 tttccccctc cctattgcca cggcggaact catcgccgcc tgccttgccc gctgctggac 11700 aggggctcgg ctgttgggca ctgacaattc cgtggtgttg tcggggaaat catcgtcctt 11760 tccttggctg ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct tctgctacgt 11820 cccttcggcc ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc 11880 tcttccgcgt cttcgccttc gccctcagac gagtcggatc tccctttggg ccgcctcccc 11940 gcatcgatac cgtcgtccac gagggaatta attcgagctc cgtaccttta agaccaatga 12000 cttacaaggc agctgtagat cttagccact ttttaaaaga aaagggggga ctggaagggc 12060 taattcactc ccaacgaaga caagatctgc tttttgcttg tactgggtct ctctggttag 12120 accagatctg agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat 12180 aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact 12240 agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagcaa 12289 <210 > 2 <211> 10709 <212> DNA <213> Artificial Sequence <220> <223> Plasmid psPAX2 <400> 2 agccattgcc ttttatggta atcgtgcgag agggcgcagg gacttccttt gtcccaaatc 60 tgtgcggagc cgaaatctgg gaggcgccgc cgcaccccct ctagcgggcg cggggcgaag 120 cggtgcggcg ccggcaggaa ggaaatgggc ggggagggcc ttcgtgcgtc gccgcgccgc 180 cgtccccttc tccctctcca gcctcggggc tgtccgcggg gggacggctg ccttcggggg 240 ggacggggca gggcggggtt cggcttctgg cgtgtgaccg gcggctctag agcctctgct 300 aaccatgttc atgccttctt ctttttccta cagctcctgg gcaacgtgct ggttattgtg 360 ctgtctcatc attttggcaa agaattcggg ccgcgttgac gcgcacggca agaggcgagg 420 ggcggcgact ggtgagagat gggtgcgaga gcgtcagtat taagcggggg agaattagat 480 cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa aatataaatt aaaacatata 540 gtatgggcaa gcagggagct agaacgattc gcagttaatc ctggcctgtt agaaacatca 600 gaaggctgta gacaaatact gggacagcta caaccatccc ttcagacagg atcagaagaa 660 cttagatcat tatataatac agtagcaacc ctctattgtg tgcatcaaag gatagagata 720 aaagacacca aggaagcttt agacaagata gaggaagagc aaaacaaaag taagaaaaaa 780 gccatccc agcaag cagcagctga cacaggacac agcaatcagg tcagccaaaa ttaccctata 840 gtgcagaaca tccaggggca aatggtacat caggccatat cacctagaac tttaaatgca 900 tgggtaaaag tagtagaaga gaaggctttc agcccagaag tgatacccat gttttcagca 960 ttatcagaag gagccacccc acaagattta aacaccatgc taaacacagt ggggggacat 1020 caagcagcca tgcaaatgtt aaaagagacc atcaatgagg aagctgcaga atgggataga 1080 gtgcatccag tgcatgcagg gcctattgca ccaggccaga tgagagaacc aaggggaagt 1140 gacatagcag gaactactag tacccttcag gaacaaatag gatggatgac acataatcca 1200 cctatcccag taggagaaat ctataaaaga tggataatcc tgggattaaa taaaatagta 1260 agaatgtata gccctaccag cattctggac ataagacaag gaccaaagga accctttaga 1320 gactatgtag accgattcta taaaactcta agagccgagc aagcttcaca agaggtaaaa 1380 aattggatga cagaaacctt gttggtccaa aatgcgaacc cagattgtaa gactatttta 1440 aaagcattgg gaccaggagc gacactagaa gaaatgatga cagcatgtca gggagtgggg 1500 ggacccggcc ataaagcaag agttttggct gaagcaatga gccaagtaac aaatccagct 1560 accataatga tacagaaagg caattttagg aaccaaagaa agactgttaa gtgtttcaat 1620 tgtggcaaag aagggcacat agccaaaaat tgcagggccc ctaggaaaaa gggctgttgg 1680 aaatgtggaa aggaaggaca ccaaatgaaa gattgtactg agagacaggc taatttttta 1740 gggaagatct ggccttccca caagggaagg ccagggaatt ttcttcagag cagaccagag 1800 ccaacagccc caccagaaga gagcttcagg tttggggaag agacaacaac tccctctcag 1860 aagcag gagc cgatagacaa ggaactgtat cctttagctt ccctcagatc actctttggc 1920 agcgacccct cgtcacaata aagatagggg ggcaattaaa ggaagctcta ttagatacag 1980 gagcagatga tacagtatta gaagaaatga atttgccagg aagatggaaa ccaaaaatga 2040 tagggggaat tggaggtttt atcaaagtaa gacagtatga tcagatactc atagaaatct 2100 gcggacataa agctataggt acagtattag taggacctac acctgtcaac ataattggaa 2160 gaaatctgtt gactcagatt ggctgcactt taaattttcc cattagtcct attgagactg 2220 taccagtaaa attaaagcca ggaatggatg gcccaaaagt taaacaatgg ccattgacag 2280 aagaaaaaat aaaagcatta gtagaaattt gtacagaaat ggaaaaggaa ggaaaaattt 2340 caaaaattgg gcctgaaaat ccatacaata ctccagtatt tgccataaag aaaaaagaca 2400 gtactaaatg gagaaaatta gtagatttca gagaacttaa taagagaact caagatttct 2460 gggaagttca attaggaata ccacatcctg cagggttaaa acagaaaaaa tcagtaacag 2520 tactggatgt gggcgatgca tatttttcag ttcccttaga taaagacttc aggaagtata 2580 ctgcatttac catacctagt ataaacaatg agacaccagg gattagatat cagtacaatg 2640 tgcttccaca gggatggaaa ggatcaccag caatattcca gtgtagcatg acaaaaatct 2700 tagagccttt t agaaaacaa aatccagaca tagtcatcta tcaatacatg gatgatttgt 2760 atgtaggatc tgacttagaa atagggcagc atagaacaaa aatagaggaa ctgagacaac 2820 atctgttgag gtggggattt accacaccag acaaaaaaca tcagaaagaa cctccattcc 2880 tttggatggg ttatgaactc catcctgata aatggacagt acagcctata gtgctgccag 2940 aaaaggacag ctggactgtc aatgacatac agaaattagt gggaaaattg aattgggcaa 3000 gtcagattta tgcagggatt aaagtaaggc aattatgtaa acttcttagg ggaaccaaag 3060 cactaacaga agtagtacca ctaacagaag aagcagagct agaactggca gaaaacaggg 3120 agattctaaa agaaccggta catggagtgt attatgaccc atcaaaagac ttaatagcag 3180 aaatacagaa gcaggggcaa ggccaatgga catatcaaat ttatcaagag ccatttaaaa 3240 atctgaaaac aggaaagtat gcaagaatga agggtgccca cactaatgat gtgaaacaat 3300 taacagaggc agtacaaaaa atagccacag aaagcatagt aatatgggga aagactccta 3360 aatttaaatt acccatacaa aaggaaacat gggaagcatg gtggacagag tattggcaag 3420 ccacctggat tcctgagtgg gagtttgtca atacccctcc cttagtgaag ttatggtacc 3480 agttagagaa agaacccata ataggagcag aaactttcta tgtagatggg gcagccaata 3540 gggaaactaa attagga aaa gcaggatatg taactgacag aggaagacaa aaagttgtcc 3600 ccctaacgga cacaacaaat cagaagactg agttacaagc aattcatcta gctttgcagg 3660 attcgggatt agaagtaaac atagtgacag actcacaata tgcattggga atcattcaag 3720 cacaaccaga taagagtgaa tcagagttag tcagtcaaat aatagagcag ttaataaaaa 3780 aggaaaaagt ctacctggca tgggtaccag cacacaaagg aattggagga aatgaacaag 3840 tagataaatt ggtcagtgct ggaatcagga aagtactatt tttagatgga atagataagg 3900 cccaagaaga acatgagaaa tatcacagta attggagagc aatggctagt gattttaacc 3960 taccacctgt agtagcaaaa gaaatagtag ccagctgtga taaatgtcag ctaaaagggg 4020 aagccatgca tggacaagta gactgtagcc caggaatatg gcagctagat tgtacacatt 4080 tagaaggaaa agttatcttg gtagcagttc atgtagccag tggatatata gaagcagaag 4140 taattccagc agagacaggg caagaaacag catacttcct cttaaaatta gcaggaagat 4200 ggccagtaaa aacagtacat acagacaatg gcagcaattt caccagtact acagttaagg 4260 ccgcctgttg gtgggcgggg atcaagcagg aatttggcat tccctacaat ccccaaagtc 4320 aaggagtaat agaatctatg aataaagaat taaagaaaat tataggacag gtaagagatc 4380 aggctgaaca tcttaagaca gc agtacaaa tggcagtatt catccacaat tttaaaagaa 4440 aaggggggat tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca 4500 tacaaactaa agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca 4560 gggacagcag agatccagtt tggaaaggac cagcaaagct cctctggaaa ggtgaagggg 4620 cagtagtaat acaagataat agtgacataa aagtagtgcc aagaagaaaa gcaaagatca 4680 tcagggatta tggaaaacag atggcaggtg atgattgtgt ggcaagtaga caggatgagg 4740 attaacacat ggaattctgc aacaactgct gtttatccat ttcagaattg ggtgtcgaca 4800 tagcagaata ggcgttactc gacagaggag agcaagaaat ggagccagta gatcctagac 4860 tagagccctg gaagcatcca ggaagtcagc ctaaaactgc ttgtaccaat tgctattgta 4920 aaaagtgttg ctttcattgc caagtttgtt tcatgacaaa agccttaggc atctcctatg 4980 gcaggaagaa gcggagacag cgacgaagag ctcatcagaa cagtcagact catcaagctt 5040 ctctatcaaa gcagtaagta gtacatgtaa tgcaacctat aatagtagca atagtagcat 5100 tagtagtagc aataataata gcaatagttg tgtggtccat agtaatcata gaatatagga 5160 aaatggccgc tgatcttcag acctggagga ggagatatga gggacaattg gagaagtgaa 5220 ttatataaat ataaagtagt aaaaattg aa ccattaggag tagcacccac caaggcaaag 5280 agaagagtgg tgcagagaga aaaaagagca gtgggaatag gagctttgtt ccttgggttc 5340 ttgggagcag caggaagcac tatgggcgca gcgtcaatga cgctgacggt acaggccaga 5400 caattattgt ctggtatagt gcagcagcag aacaatttgc tgagggctat tgaggcgcaa 5460 cagcatctgt tgcaactcac agtctggggc atcaagcagc tccaggcaag aatcctggct 5520 gtggaaagat acctaaagga tcaacagctc ctggggattt ggggttgctc tggaaaactc 5580 atttgcacca ctgctgtgcc ttggaatgct agttggagta ataaatctct ggaacagatt 5640 tggaatcaca cgacctggat ggagtgggac agagaaatta acaattacac aagcttaata 5700 cactccttaa ttgaagaatc gcaaaaccag caagaaaaga atgaacaaga attattggaa 5760 ttagataaat gggcaagttt gtggaattgg tttaacataa caaattggct gtggtatata 5820 aaattattca taatgatagt aggaggcttg gtaggtttaa gaatagtttt tgctgtactt 5880 tctatagtga atagagttag gcagggatat tcaccattat cgtttcagac ccacctccca 5940 accccgaggg gacccgacag gcccgaagga atagaagaag aaggtggaga gagagacaga 6000 gacagatcca ttcgattagt gaacggatcc ttggcactta tctgggacga tctgcggagc 6060 ctgtgcctct tcagctacca ccgcttgaga gac ttactct tgattgtaac gaggattgtg 6120 gaacttctgg gacgcagggg gtgggaagcc ctcaaatatt ggtggaatct cctacaatat 6180 tggagtcagg agctaaagaa tagtgctgtt agcttgctca atgccacagc catagcagta 6240 gctgagggga cagatagggt tatagaagta gtacaaggag cttgtagagc tattcgccac 6300 atacctagaa gaataagaca gggcttggaa aggattttgc tataagctcg aaacaaccgg 6360 tacctctaga actatagcta gcagatcttt ttccctctgc caaaaattat ggggacatca 6420 tgaagcccct tgagcatctg acttctggct aataaaggaa atttattttc attgcaatag 6480 tgtgttggaa ttttttgtgt ctctcactcg gaaggacata tgggagggca aatcatttaa 6540 aacatcagaa tgagtatttg gtttagagtt tggcaacata tgcccatatg ctggctgcca 6600 tgaacaaagg ttggctataa agaggtcatc agtatatgaa acagccccct gctgtccatt 6660 ccttattcca tagaaaagcc ttgacttgag gttagatttt ttttatattt tgttttgtgt 6720 tatttttttc tttaacatcc ctaaaatttt ccttacatgt tttactagcc agatttttcc 6780 tcctctcctg actactccca gtcatagctg tccctcttct cttatggaga tccctcgacc 6840 tgcagcccaa gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg 6900 ctcacaattc cacacaacat acgagccgga agcataaag t gtaaagcctg gggtgcctaa 6960 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac 7020 ctgtcgtgcc agcggatccg catctcaatt agtcagcaac catagtcccg cccctaactc 7080 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa 7140 ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt 7200 gaggaggctt ttttggaggc ctaggctttt gcaaaaagct aacttgttta ttgcagctta 7260 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact 7320 gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct ggatccgctg 7380 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct 7440 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac 7500 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga 7560 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat 7620 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac 7680 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct 7740 gttccgaccc tgccgcttac cggatacctg tccgcctttc tccc ttcggg aagcgtggcg 7800 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg 7860 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt 7920 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg 7980 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac 8040 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga 8100 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt 8160 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt 8220 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga 8280 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc 8340 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct 8400 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata 8460 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca 8520 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga 8580 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga 8640 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg 8700 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga 8760 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt 8820 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct 8880 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca 8940 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat 9000 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga 9060 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc 9120 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg 9180 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc 9240 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt 9300 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca 9360 cctgggtcga cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt 9420 tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcct ggctg 9480 accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc 9540 aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc 9600 agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg 9660 gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat 9720 ctacgtatta gtcatcgcta ttaccatggt cgaggtgagc cccacgttct gcttcactct 9780 ccccatctcc cccccctccc cacccccaat tttgtattta tttatttttt aattattttg 9840 tgcagcgatg ggggcggggg gggggggggc gcgcgccagg cggggcgggg cggggcgagg 9900 ggcggggcgg ggcgaggcgg agaggtgcgg cggcagccaa tcagagcggc gcgctccgaa 9960 agtttccttt tatggcgagg cggcggcggc ggcggcccta taaaaagcga agcgcgcggc 10020 gggcgggagt cgctgcgcgc tgccttcgcc ccgtgccccg ctccgccgcc gcctcgcgcc 10080 gcccgccccg gctctgactg accgcgttac tcccacaggt gagcgggcgg gacggccctt 10140 ctcctccggg ctgtaattag cgcttggttt aatgacggct tgtttctttt ctgtggctgc 10200 gtgaaagcct tgaggggctc cgggagggcc ctttgtgcgg ggggagcggc tcggggggtg 10260 cgtgcgtgtg tgtgtgcgtg gggagcgccg cgtgcggctc cgcgctgccc ggcggc tgtg 10320 agcgctgcgg gcgcggcgcg gggctttgtg cgctccgcag tgtgcgcgag gggagcgcgg 10380 ccgggggcgg tgccccgcgg tgcggggggg gctgcgaggg gaacaaaggc tgcgtgcggg 10440 gtgtgtgcgt gggggggtga gcagggggtg tgggcgcgtc ggtcgggctg caaccccccc 10500 tgcacccccc tccccgagtt gctgagcacg gcccggcttc gggtgcgggg ctccgtacgg 10560 ggcgtggcgc ggggctcgcc gtgccgggcg gggggtggcg gcaggtgggg gtgccgggcg 10620 gggcggggcc gcctcgggcc ggggagggct cgggggaggg gcgcggcggc ccccggagcg 10680 ccggcggctg tcgaggcgcg gcgagccgc 10709 <210 > 3 <211> 9202 <212> DNA <213> Artificial Sequence <220> <223> SARS-CoV-2 Spike plasmid <400> 3 ccgacaattg catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac 60 gggta cc tac tacgcgttga cattagtta ttag t cc tca tacgcgttga cattgattat tggt ataacttacg gtaaatggcc 180 cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca 240 tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattattta cggtaaactg tgccctattagt gtcaatg tgccctattagtac gtatcatag 360 acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt 420 ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca 480 tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg 540 tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact 600 ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag 660 ctctctggct aactagagaa cccactgctt actggcttat cgaaattaat acgactcact 720 atagggagac ccaagctggc tagcatgttt gtcttcctgg tcctgctgcc tctggtctcg 780 tctcagtgcg tgaacctgac tactagaacc cagctgcctc ctgcctatac taactccttc 840 acccgcggcg tgtactaccc agacaaggtg ttccgcagct ccgtgctgca ctccacccag 900 gatctgttcc tgcccttctt cagcaacgtg acctggttcc acgccatcca cgtgagcggc 960 accaatggca ccaagcggtt cgacaatccc gtgctgccat tcaacgatgg cgtgtacttc 1020 gcctccaccg agaagagcaa catcatccgc ggctggatct tcggcaccac cctggactcc 1080 aagacccaga gcctgctgat cgtgaacaat gccaccaacg tggtcatcaa ggtgtgcgag 1140 ttccagttct gcaatgatcc attcctgggc gtgtactacc acaagaacaa taagtcctgg 1200 atggagagcg agttc cgcgt gtacagctcc gccaacaatt gcaccttcga gtacgtgtcc 1260 cagcccttcc tgatggacct ggagggcaag cagggcaatt tcaagaacct gcgcgagttc 1320 gtgttcaaga atatcgatgg ctacttcaag atctactcca agcacacccc catcaacctg 1380 gtgcgcgacc tgccacaggg cttcagcgcc ctggagccac tggtggatct gccaatcggc 1440 atcaacatca ccaggttcca gaccctgctg gccctgcacc gcagctacct gaccccaggc 1500 gacagctcca gcggatggac cgctggagct gctgcctact acgtgggcta cctgcagccc 1560 cgcaccttcc tgctgaagta caacgagaat ggcaccatca ccgacgccgt ggattgcgcc 1620 ctggatccac tgtccgagac aaagtgcacc ctgaagagct tcaccgtgga gaagggcatc 1680 taccagacct ccaatttccg cgtgcagcca accgagagca tcgtgcgctt ccccaatatc 1740 accaacctgt gcccattcgg cgaggtgttc aacgctacca ggttcgccag cgtgtacgct 1800 tggaatcgca agcgcatctc caactgcgtg gccgactaca gcgtgctgta caactccgcc 1860 agcttctcca ccttcaagtg ctacggcgtg tcccccacca agctgaatga tctgtgcttc 1920 accaacgtgt acgccgatag cttcgtgatc aggggcgacg aggtgcgcca gatcgctcca 1980 ggacagaccg gcaagatcgc tgactacaat tacaagctgc ccgacgattt caccggctgc 2040 gtgatcgcct ggaactccaa caatctggat agcaaagtgg gcggcaacta caattacctg 2100 taccgcctgt tccgcaagtc caatctgaag ccattcgagc gcgacatctc caccgagatc 2160 taccaggctg gaagcacccc atgcaatgga gtggagggct tcaactgcta cttccccctg 2220 cagagctacg gcttccagcc aaccaacgga gtgggatacc agccatacag ggtggtggtg 2280 ctgtccttcg agctgctgca cgctccagct accgtgtgcg gaccaaagaa gagcaccaat 2340 ctggtgaaga acaagtgcgt gaacttcaat ttcaacggcc tgaccggaac cggcgtgctg 2400 accgagtcca acaagaagtt cctgccattc cagcagttcg gaagggacat cgctgatacc 2460 accgacgccg tgcgcgaccc acagaccctg gagatcctgg atatcacccc atgctccttc 2520 ggcggcgtga gcgtgatcac cccaggaacc aataccagca accaggtggc cgtgctgtac 2580 caggacgtga attgcaccga ggtgccagtg gctatccacg ctgatcagct gaccccaacc 2640 tggcgcgtgt acagcaccgg atccaacgtg ttccagaccc gcgccggatg cctgatcgga 2700 gctgagcacg tgaacaattc ctacgagtgc gacatcccaa tcggagctgg aatctgcgcc 2760 agctaccaga cccagaccaa ctccccaagg agggctcgca gcgtggccag ccagtccatc 2820 atcgcctaca ccatgtccct gggcgccgag aatagcgtgg cctacagcaa caattccatc 2880 gccatcccaa ccaacttcac catctc cgtg accaccgaga tcctgcccgt gtccatgacc 2940 aagaccagcg tggactgcac catgtacatc tgcggcgatt ccaccgagtg cagcaacctg 3000 ctgctgcagt acggcagctt ctgcacccag ctgaatcgcg ccctgaccgg aatcgctgtg 3060 gagcaggata agaacaccca ggaggtgttc gcccaggtga agcagatcta caagaccccc 3120 ccaatcaagg acttcggcgg cttcaatttc agccagatcc tgcccgatcc aagcaagccc 3180 tccaagcgca gcttcatcga ggacctgctg ttcaacaagg tgaccctggc cgatgccggc 3240 ttcatcaagc agtacggcga ttgcctgggc gacatcgctg cccgcgacct gatctgcgcc 3300 cagaagttca atggcctgac cgtgctgcca ccactgctga ccgatgagat gatcgctcag 3360 tacacctccg ccctgctggc cggaaccatc accagcggat ggaccttcgg cgctggagcc 3420 gccctgcaga tccccttcgc catgcagatg gcctaccgct tcaacggcat cggcgtgacc 3480 cagaatgtgc tgtacgagaa ccagaagctg atcgccaatc agttcaactc cgccatcggc 3540 aagatccagg actccctgtc cagcaccgcc agcgccctgg gcaagctgca ggatgtggtg 3600 aatcagaacg cccaggccct gaataccctg gtgaagcagc tgtccagcaa cttcggcgcc 3660 atctccagcg tgctgaatga tatcctgagc cgcctggaca aggtggaggc tgaggtgcag 3720 atcgataggc tgatcaccgg ccgcctgcag t ccctgcaga cctacgtgac ccagcagctg 3780 atcagggctg ctgagatcag ggccagcgcc aatctggctg ctaccaagat gtccgagtgc 3840 gtgctgggac agagcaagag ggtggacttc gtgc ggcaaggggtcgt agcc tggacttc gtgc ggcaagg ggtacct acct aagaacttca ccaccgctcc agctatctgc cacgatggca aggctcactt cccacgcgag 4020 ggcgtgttcg tgtccaacgg cacccactgg ttcgtgaccc agcgcaattt ctacgagccc 4080 cagatcatca ccaccgacaa taccttcgtg agcggcaact gcgacgtggt catcggaatc 4140 gtgaacaata ccgtgtacga tcccctgcag ccagagctgg actccttcaa ggaggagctg 4200 gataagtact tcaagaatca caccagcccc gacgtggatc tgggcgacat ctccggcatc 4260 aatgccagcg tggtgaacat ccagaaggag atcgaccgcc tgaacgaggt ggccaagaat 4320 ctgaacgagt ccctgatcga tctgcaggag ctgggcaagt acgagcagta catcaagtgg 4380 ccatggtaca tctggctggg cttcatcgcc ggcctgatcg ccatcgtgat ggtgaccatc 4440 atgctgtgct gcatgacctc ctgctgcagc tgcctgaagg gctgctgctc ctgcggcagc 4500 tgctgcaagt tcgatgagga cgatagcgag cccgtgctga agggcgtcaa actgcactat 4560 acaggctcca ccgagacatc ccaggtcgct cccgcttagc tcgagtctag agggcccgtt 4620 taaacccgct gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc 4680 tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat 4740 gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg 4800 caggac agca agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc 4860 tctatggctt ctgaggcgga aagaaccagc tggggctcta gggggtatcc ccacgcgccc 4920 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt 4980 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc 5040 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta 5100 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc 5160 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg 5220 ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt 5280 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat 5340 taattctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca 5400 gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct 5460 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc 5520 ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg 5580 gctgactaat tttttttatt tatgcagagg ccgaggccgc ctctgcctct gagctattcc 5640 agaagtagtg a ggaggcttt tttggaggcc taggcttttg caaaaagctc ccgggagctt 5700 gtatatccat tttcggatct gatcaagaga caggatgagg atcgtttcgc atgattgaac 5760 aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact 5820 gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc 5880 gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg caggacgagg 5940 cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg 6000 tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt 6060 catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc 6120 atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag 6180 cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg 6240 ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc 6300 tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt 6360 ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg 6420 ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt 6480 acggtatcgc cgctccc gat tcgcagcgca tcgccttcta tcgccttctt gacgagttct 6540 tctgagcggg actctggggt tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg 6600 agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga 6660 cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccccaa 6720 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa 6780 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta 6840 tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt 6900 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa 6960 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact 7020 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc 7080 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg 7140 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc 7200 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag 7260 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca 7320 tcacaaaaat cgacgctcaa gt cagaggtg gcgaaacccg acaggactat aaagatacca 7380 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg 7440 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag 7500 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt 7560 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca 7620 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg 7680 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt 7740 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc 7800 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg 7860 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg 7920 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta 7980 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg 8040 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg 8100 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc 8160 atctggcccc agtgctgcaa tgataccg cg agacccacgc tcaccggctc cagatttatc 8220 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc 8280 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag 8340 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat 8400 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg 8460 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt 8520 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag 8580 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg 8640 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt 8700 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct 8760 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac 8820 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat 8880 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat 8940 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca 9000 aataggggtt ccgcgcacat ttccccgaaa agt gccacct gacgtcgacg gatcgggaga 9060 tctcccgatc ccctatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc 9120 cagtatacacgc tccctgcttg tgttgttggag gtcgctgaaggt agtgcggc

Claims (13)

A pharmaceutical composition for preventing or treating infection with enveloped viruses, comprising Platycodin D as an active ingredient. The method of claim 1,
The envelope virus is a severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), Middle East Respiratory Syndrome (MERS) virus, severe acute respiratory syndrome (Severe Acute Respiratory Syndrome, SARS) any of the virus A pharmaceutical composition for preventing or treating an enveloped virus infection, characterized in that there is only one. The method of claim 1,
The Platycodin D (Platycodin D) is a pharmaceutical composition for preventing or treating an enveloped virus infection, which inhibits the entry into cells of the enveloped virus and / or the binding of the enveloped virus to the spike protein. . The method of claim 1,
The Platycodin D (Platycodin D) will be included in 0.001 to 50% by weight based on the total weight of the composition, a pharmaceutical composition for preventing or treating an enveloped virus infection. Gilgyeong ( Platycodon grandiflorum ) A pharmaceutical composition for preventing or treating an enveloped virus infection comprising an extract. 6. The method of claim 5,
The Gilgyeong ( Platycodon grandiflorum ) extract inhibits cell entry of the enveloped virus and / or inhibits binding to the spike protein of the enveloped virus and / or inhibits cleavage of the coronavirus spike protein during intracellular viral packaging of the enveloped virus A pharmaceutical composition for preventing or treating infection with enveloped viruses. 6. The method of claim 5,
The Gilgyeong ( Platycodon grandiflorum ) extract is contained in an amount of 0.001 to 50% by weight based on the total weight of the composition, a pharmaceutical composition for preventing or treating an enveloped virus infection. A pharmaceutical preparation comprising the pharmaceutical composition for preventing or treating an enveloped virus infection according to any one of claims 1 to 7. 9. The method of claim 8,
The pharmaceutical formulation is a solid dosage form, a spray dosage form, or a liquid dosage form. 9. The method of claim 8,
The pharmaceutical formulation is for oral, parenteral, intranasal, oral, oral, sublingual, airway spray or rectal administration. 9. The method of claim 8,
The pharmaceutical preparation is a pharmaceutical preparation in the form of capsules, pills, tablets, solutions, suspensions, sprays, foams, patches or pastes. Gilkyung ( Platycodon grandiflorum ) Extract and / or Platycodin D (Platycodin D) containing the enveloped virus (enveloped viruses) infection prevention or improvement food composition. 12. The method of claim 11,
The Gilkyung ( Platycodon grandiflorum ) extract, Platycodin D (Platycodin D) or a mixture thereof is included in an amount of 0.001 to 50% by weight based on the total weight of the composition. Food for preventing or improving infection with enveloped viruses composition.

Images