COMPOSITION FOR NEUTRALIZING CORONAVIRUS

Abstract

The present disclosure relates to a novel protein that specifically binds to a coronavirus, and a composition for neutralizing a coronavirus. A novel protein provided in the present disclosure is a novel protein in which virus binding ability is maintained but toxicity is eliminated on the basis of virus binding properties of bean-derived protein lectins, and thus can be used as a material for preventing the infection of a coronavirus or alleviating infectious diseases.

Claims

1. A composition for neutralizing a coronavirus, wherein the composition comprises a protein comprising amino acids of the following sequence as an active ingredient. TABLE-US-00005 ELDTYPNTDIGDPSYPHIGIDIKSVRSKKTAKWNMQDGKVGTAHIIYNS VDKRLSAVVSYPNADATSVSYDVDLNDVLPEWVRVGLSASTGLYKETNT ILSWSFTSKLKSNSTHQTDALHFMFNQFSKDQKDLILQGDATTGTDGNL ELTRVSSNGSPEGSSVGRALFYAPVHIWESSATVSAFEATFAFLIK

2. The composition of claim 1, wherein the protein is derived from bean-derived lectin protein.

3. The composition of claim 1, wherein the protein is extracted from fermented Canavalia ensiformis.

4. The composition of claim 1, wherein, for the protein, an amino acid of a lectin toxic portion is deleted from ConcanavalinA derived from Canavalia ensiformis.

5. The composition of claim 4, wherein the amino acid of the lectin toxic portion comprises 1.sup.st to 7.sup.th amino acids and amino acids after a 200.sup.th amino acid in ConcanavalinA.

6. A pharmaceutical composition comprising the composition for neutralizing a coronavirus of claim 1 as an active ingredient.

7. A food composition comprising the composition for neutralizing a coronavirus of claim 1 as an active ingredient.

8. A health functional food composition comprising the composition for neutralizing a coronavirus of claim 1 as an active ingredient.

9. A quasi-drug composition comprising the composition for neutralizing a coronavirus of claim 1 as an active ingredient.

10. A composition for adding feed, the composition comprising the composition for neutralizing a coronavirus of claim 1 as an active ingredient.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0040] FIG. 1 shows the results of isolation of proteins obtained from fermented Canavalia ensiformis using LC.

[0041] FIG. 2 shows the results of mass value information for peptides identified from fermented Canavalia ensiformis.

[0042] FIG. 3 shows the results of ETD amino acid sequencing of the protein as an active sub stance.

[0043] FIG. 4 shows the mass spectrometry spectrum and deconvolution results for tConA.

[0044] FIG. 5 is a graph showing the human coronavirus neutralizing ability of tConA.

[0045] FIG. 6 shows a verification image of the human coronavirus neutralizing ability of tConA.

[0046] FIG. 7 shows the IC50 values of tConA for neutralizing a coronavirus.

[0047] FIG. 8 is a comparison of the cytokine production ability of splenocytes of ConA and tConA.

BEST MODE FOR CARRYING OUT THE INVENTION

Example 1. Obtaining Raw Materials Through Fermenting Canavalia ensiformis

[0048] 200 ml (1% of tryptone, 0.5% of yeast extract, and 1% of sodium chloride) of LB broth miller (LBL407.500, Bioshop, Canada) containing tryptone, yeast extract and sodium chloride was additionally added with 8.25 g of Canavalia ensiformis powder, inoculated with a strain corresponding to 5% (v/v) of a medium, and then fermented for 7 days by stirring at 33° C. The fermented supernatant was centrifuged at 12,000 rpm, 4° C. for 20 minutes, the supernatant was collected, the residue was fractionated using filter paper (Whatman filter paper 4, 25 nm), and the filtrate was lyophilized by removing salts using a 10 kDa membrane filter (Sartorius 7554-95, MASTERFLEX, USA) and concentrating the fermented liquid capacity by 2.4 times. The obtained solid content was the final product of the fermented Canavalia ensiformis, and SDS-PAGE was performed to check the fermentation termination time.

[0049] From day 0 to day 7 of fermentation, 1 mg of each sample of the fermented Canavalia ensiformis was taken and dissolved in 1 ml of distilled water and subjected to SDS-PAGE analysis through 15% of polyacrylamide gel. A major band was observed at 48 kDa molecular weights on day 0 of fermentation. However, from day 1 of fermentation, the band with a molecular weight of 48 kDa was decreased, and the band of 25 kDa or less, the size of ConA, was increased. By day 7, a major band was observed more clearly at molecular weights of 25 kDa or less.

[0050] As a result of isolation by size using 15% SDS-PAGE, a protein of about 48 kDa exists as the main protein on day 0 of fermentation, and three types of protein of about 20 to 25 kDa were produced when fermented for 4 days using Bacillus subtillis natto. Through peptide coverage analysis, specific homology was identified from the standard material, ConA, and the fermented Canavalia ensiformis. After cutting the FO band of Canavalia ensiformis powder (column 2 in FIG. 2) and the F2, F3 and F4 bands of fermented Canavalia ensiformis (column 3 in FIG. 2), in-gel digestion was performed to wash with 25 mM ABC in 50% ACN solution. After decomposition into peptides with trypsin gold (Promega, V5280) under 50 mM ABC conditions, desalting was performed using Oasis SPE (Waters, 186001828BA), and peptide analysis was performed using nanoUPLC-Synapt G2 si (Waters, USA) equipment. The results obtained through LC-MS were analyzed using PLGS (ProteinLynx Global Server (version 3.0, PLGS, Waters). As a result, in the case of FO band, CVJB concanavalinA Jack bean (UniProtKB: CVJB Con A, gi72333) expected to be a commercial ConA sequence and 97.5% of homology were identified, and in the case of F2, F3, and F4 bands, CVJB concanavalinA, Jack bean (UniProtKB: CVJB Con A, gi72333) and each sequence coverage were identified as 81.4%, 53.2%, and 62.86%.

TABLE-US-00001 TABLE 1 Sequence Band Access Same cover name number Description Score peptide (%) F0 gi72333 CVJB concanavalinA- 2389.3 23 97.5 jack bean F2 gi72333 CVJB concanavalinA- 730.1 9 81.4 jack bean F3 gi72333 CVJB concanavalinA- 629.7 8 53.2 jack bean F4 gi72333 CVJB concanavalinA- 1341.8 8 62.86 jack bean

Example 2. Active Ingredient Identification

[0051] In order to measure the exact molecular weight of the proteins isolated from the fermented Canavalia ensiformis, the intact protein mass values were calculated for the main band on the day 0 of fermentation of Canavalia ensiformis and the protein concentrate with a molecular weight of 30 KDa or less, which was identified in the fermented product on day 4 of fermentation of Canavalia ensiformis. Molecular weight measurement was performed using nano ultra-high performance liquid chromatography (UPLC, ACQUITY UPLC I-Class/SYNAPT G2-S HDMS, Waters) for fermentation analysis, and the analysis conditions are as shown below.

[0052] An ACQUITYBEH300 C18 (1.7 μm x 2.1×50 mm) column was used, a column temperature at 60° C., mobile phase solvent A (0.1% of formic acid in water), and solvent B (0.1% of formic acid in acetonitrile) were used. The mass spectrometer performed analysis in the ESI positive mode, and the analysis conditions were set to a capillary voltage of 3.0 kV, a cone voltage of 30 V, a source temperature of 120° C., and a scan time of 0.5 seconds. ESI prot 1.0 was calculated and the deconvoluted MW (Da) and std values as shown in Table 2 were set.

[0053] For the F2, F3 and F4 bands, as CVJB concanavalinA, Jack bean (UniProtKB: CVJB Con A, gi72333) and each sequence coverage were identified as 81.4%, 53.2%, and 62.86%, due to the nature of trypsin, which recognizes lysine and arginine residues, the exact cleavage position may not be checked. However, F2 was predicted to be the protein (26213.96 Da) of the F2 region of FIG. 4 identified when the mass value of the intact protein was measured with ConA in a monomer state. As a result of calculating the mass value of the F3 band, 21175.48 Da was predicted to be the protein (21176 Da) of the F3 region of FIG. 3 identified when the mass value of the intact protein was measured. In the case of the F4 band, as the mass value was calculated as 18796.86 Da, it was predicted to be the protein (18796.8 Da) of the F4 region of FIG. 3 identified when the mass value of the intact protein was measured (FIG. 1 and Table 2).

TABLE-US-00002 TABLE 2 Charge (+) Peak (m/z) MW(Da) Deconvoluted MW (Da) F2 29 904.9487 26214.2820 26213.9637 ± 0.7529 28 937.2229 26214.0188 27 971.8983 26214.0397 26 1009.2819 26215.1229 25 1049.5328 26213.1215 24 1093.2675 26214.2294 23 1140.7080 26213.1019 22 1192.5895 26214.7943 21 1249.2468 26213.0160 20 1311.7194 26214.2292 19 1380.5923 26212.1028 18 1457.2847 26212.9816 17 1543.1046 26215.64332 F3 28 757.2388 21174.4640 21175.4820 ± 0.9101 27 785.3299 21176.6929 26 815.4147 21174.5783 25 848.0338 21175.6465 24 883.3274 21175.6670 23 921.6443 21174.6362 22 963.4954 21174.7241 21 1009.4067 21176.3739 20 1059.8358 21176.5572 19 1121.5699 21290.6772 18 1177.6122 21178.8766 17 1246.6261 21175.5087

Example 3. Sequence Analysis of Fermented Canavalia ensiformis

[0054] The protein sequence of the fermented Canavalia ensiformis obtained above was analyzed. ETD was used for F3 among fermentation proteins. As a result of identifying the amino acid sequence, it was found to be a novel protein in which some amino acids were deleted compared to the known protein ConcanavalinA. From these results, it was assumed that some of the amino acids were cleaved during the fermentation process, and the novel protein was named truncated ConcanavalinA (tConA).

TABLE-US-00003 TABLE 3 tConA ConA ELDTYPNTDIGDPSYPHI ADTIVAVELDTYPNTD GIDIKSVRSKKTAKWNMQ IGDPSYPHIGIDIKSV DGKVGTAHIIYNSVDKRL RSKKTAKWNMQDGKVG SAVVSYPNADATSVSYDV TAHIIYNSVDKRLSAV DLNDVLPEWVRVGLSAST VSYPNADATSVSYDVD GLYKETNTILSWSFTSKL LNDVLPEWVRVGLSAS KSNSTHQTDALHFMFNQF TGLYKETNTILSWSFT SKDQKDLILQGDATTGTD SKLKSNSTHQTDALHF GNLELTRVSSNGSPEGSS MFNQFSKDQKDLILQG VGRALFYAPVHIWESSAT DATTGTDGNLELTRVS VSAFEATFAFLIK SNGSPEGSSVGRALFY APVHIWESSATVSAFE ATFAFLIKSPDSHPAD GIAFFISNIDSSIPSG STGRLLGLFPDAN Theoretical pI/Mw: Theoretical pI/Mw: 5.47/21133.55 5.00/25572.38 Da

Example 4. Identification of Human Coronavirus Neutralizing Power of tConA

[0055] The virus to be identified was Human Coronavirus, which was treated with 1×10{circumflex over ( )}5 GFP-transducing units/200 μl and was used. When applied to the experiment, it was applied by diluting 2 to 10 times in DMEM media in a unit of 100 ppm (50 μl). The treatment time is 1 hour and the treatment temperature is 4° C.

[0056] Virus inoculation was performed on huh-7 cells (human hepatocellular carcinoma cell line), and cells were prepared as 1×10{circumflex over ( )}5 cells/well in a 24-well plate. After washing the culture supernatant, 250 μl of the virus-tConA mixture was inoculated into Huh-7 cells. The unbound virus was washed 3 times in DEME medium supplemented with 2% FBS and 1× penicillin/streptomycin. After culturing the washed cells for 24 hours, FACS analysis was performed in DEME medium supplemented with 2% FBS and 1× penicillin/streptomycin.

[0057] At MOI=1, the percentage of infected cells is theoretically 37% based on the Poisson distribution. The percentage of infection with untreated virus ranged from 31% to 41% in repeated experiments, with a mean of 37%. Data for the untreated virus samples indicate that the testing process performed as expected. The percentage of GFP-positive cells in the virus-only sample was normalized to 100% for comparison of each independent test.

[0058] tConA was diluted 10-fold and mixed with the virus to identify the virus neutralizing ability of tConA. Inhibition of viral entry was identified up to 1.25 ppm. At 0.2 ppm, the inhibitory effect was lower than 30%, indicating that the IC50 would be between 1.25 ppm and 0.2 ppm.

[0059] In order to determine the IC50 of tConA against virus, tConA was serially diluted 2-fold and mixed with virus. Up to 2.5 ppm, the inhibitory effect was 99% or higher. The IC50 of tConA is estimated to be 0.386 ppm.

[0060] In conclusion, tConA was 99% or higher effective in inhibiting viral entry into Huh-7 cells down to 1.25 ppm. The IC50 is estimated to be 0.386 ppm.

Example 5. Identification of Toxicity Reducing Effect of tConA

[0061] After killing two normal mouse species (C57BL/6, Balb/C) with ether, the spleen was aseptically extracted to prepare a spleen cell liquid, and then 500 μl (4×10.sup.6 cells/ml) of the cell liquid was added to a 24 well flat-bottomed plate. After stabilization by culturing for 30 minutes (37° C., 5% CO2), 100 μl (2 μg/ml) of ConA and tConA and 300 μl of 10% FBS-RPMI medium were added to each well, and the culture solution obtained by culturing for 24 hours and 48 hours was used while being stored at −70° C. According to the sandwich ELISA method provided by PharMingen, the amount of cytokine (IFN-γ, IL-17, IL-4, IL-5, IL-13) production in the culture medium was compared and measured. In the case of C57BL/6, when only 48 hours results were compared, IFN-γ was reduced by 63.4% compared to ConA when treated with tConA, IL-17 was reduced by 92.3%, IL-4 was not detected, and IL-5 was reduced by 97.2%. As such, tConA maintains its antiviral ability and lowers its toxicity, so it is expected to have a wide range of applications when applied to humans or animals.

TABLE-US-00004 Sequence List Fee Text ELDTYPNTDIGDPSYPHIGIDIKSVRSKKTAKWNMQDGKVGTAHIIYNS VDKRLSAVVSYPNADATSVSYDVDLNDVLPEWVRVGLSASTGLYKETNT ILSWSFTSKLKSNSTHQTDALHFMFNQFSKDQKDLILQGDATTGTDGNL ELTRVSSNGSPEGSSVGRALFYAPVHIWESSATVSAFEATFAFLIK