KR102341805B1 - Anti-IsaA G4 antibody specific to Staphylococcus aureus and use thereof - Google Patents
Anti-IsaA G4 antibody specific to Staphylococcus aureus and use thereof Download PDFInfo
- Publication number
- KR102341805B1 KR102341805B1 KR1020190154518A KR20190154518A KR102341805B1 KR 102341805 B1 KR102341805 B1 KR 102341805B1 KR 1020190154518 A KR1020190154518 A KR 1020190154518A KR 20190154518 A KR20190154518 A KR 20190154518A KR 102341805 B1 KR102341805 B1 KR 102341805B1
- Authority
- KR
- South Korea
- Prior art keywords
- antibody
- isaa
- antigen
- binding fragment
- seq
- Prior art date
Links
- 241000191967 Staphylococcus aureus Species 0.000 title claims abstract description 29
- 239000000427 antigen Substances 0.000 claims abstract description 56
- 108091007433 antigens Proteins 0.000 claims abstract description 56
- 102000036639 antigens Human genes 0.000 claims abstract description 56
- 239000012634 fragment Substances 0.000 claims abstract description 45
- 230000002401 inhibitory effect Effects 0.000 claims abstract description 8
- 239000000203 mixture Substances 0.000 claims description 43
- 239000004480 active ingredient Substances 0.000 claims description 23
- 150000007523 nucleic acids Chemical class 0.000 claims description 12
- 239000013604 expression vector Substances 0.000 claims description 10
- 108020004707 nucleic acids Proteins 0.000 claims description 10
- 102000039446 nucleic acids Human genes 0.000 claims description 10
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 9
- 238000003259 recombinant expression Methods 0.000 claims description 8
- 230000000844 anti-bacterial effect Effects 0.000 claims description 7
- 230000004927 fusion Effects 0.000 claims description 4
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims description 3
- 125000003275 alpha amino acid group Chemical group 0.000 claims 6
- 238000000338 in vitro Methods 0.000 claims 1
- 230000012010 growth Effects 0.000 abstract description 5
- 208000012657 Atopic disease Diseases 0.000 abstract description 2
- 201000004624 Dermatitis Diseases 0.000 abstract description 2
- 238000001514 detection method Methods 0.000 abstract description 2
- 230000006872 improvement Effects 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 25
- 150000001413 amino acids Chemical group 0.000 description 19
- 239000013598 vector Substances 0.000 description 14
- 238000009472 formulation Methods 0.000 description 13
- 108090000623 proteins and genes Proteins 0.000 description 11
- 102100035360 Cerebellar degeneration-related antigen 1 Human genes 0.000 description 10
- 206010012438 Dermatitis atopic Diseases 0.000 description 9
- 238000002965 ELISA Methods 0.000 description 9
- 201000008937 atopic dermatitis Diseases 0.000 description 9
- 239000006210 lotion Substances 0.000 description 9
- 238000000034 method Methods 0.000 description 9
- 239000000243 solution Substances 0.000 description 9
- 230000036541 health Effects 0.000 description 8
- 239000008194 pharmaceutical composition Substances 0.000 description 8
- 238000004458 analytical method Methods 0.000 description 7
- 239000006071 cream Substances 0.000 description 7
- 102000004169 proteins and genes Human genes 0.000 description 7
- 241000894006 Bacteria Species 0.000 description 6
- 108020004414 DNA Proteins 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 description 6
- -1 pack Substances 0.000 description 6
- 235000018102 proteins Nutrition 0.000 description 6
- 241000588724 Escherichia coli Species 0.000 description 5
- 239000002537 cosmetic Substances 0.000 description 5
- 235000013376 functional food Nutrition 0.000 description 5
- 239000002609 medium Substances 0.000 description 5
- 239000000843 powder Substances 0.000 description 5
- 238000010206 sensitivity analysis Methods 0.000 description 5
- 210000003491 skin Anatomy 0.000 description 5
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 4
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 4
- 229930027917 kanamycin Natural products 0.000 description 4
- 229960000318 kanamycin Drugs 0.000 description 4
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 4
- 229930182823 kanamycin A Natural products 0.000 description 4
- 238000004091 panning Methods 0.000 description 4
- 230000009466 transformation Effects 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 3
- 238000012286 ELISA Assay Methods 0.000 description 3
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- 241000218588 Lactobacillus rhamnosus Species 0.000 description 3
- 108091028043 Nucleic acid sequence Proteins 0.000 description 3
- 229920001213 Polysorbate 20 Polymers 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- 239000003242 anti bacterial agent Substances 0.000 description 3
- 238000011091 antibody purification Methods 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 239000003937 drug carrier Substances 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 239000012091 fetal bovine serum Substances 0.000 description 3
- BRZYSWJRSDMWLG-CAXSIQPQSA-N geneticin Chemical compound O1C[C@@](O)(C)[C@H](NC)[C@@H](O)[C@H]1O[C@@H]1[C@@H](O)[C@H](O[C@@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](C(C)O)O2)N)[C@@H](N)C[C@H]1N BRZYSWJRSDMWLG-CAXSIQPQSA-N 0.000 description 3
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 3
- 230000003834 intracellular effect Effects 0.000 description 3
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 3
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- 239000000454 talc Substances 0.000 description 3
- 229910052623 talc Inorganic materials 0.000 description 3
- 229940124597 therapeutic agent Drugs 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 238000001262 western blot Methods 0.000 description 3
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 2
- UAIUNKRWKOVEES-UHFFFAOYSA-N 3,3',5,5'-tetramethylbenzidine Chemical compound CC1=C(N)C(C)=CC(C=2C=C(C)C(N)=C(C)C=2)=C1 UAIUNKRWKOVEES-UHFFFAOYSA-N 0.000 description 2
- 239000004475 Arginine Substances 0.000 description 2
- 108091026890 Coding region Proteins 0.000 description 2
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 2
- 102000053602 DNA Human genes 0.000 description 2
- LCGLNKUTAGEVQW-UHFFFAOYSA-N Dimethyl ether Chemical compound COC LCGLNKUTAGEVQW-UHFFFAOYSA-N 0.000 description 2
- 206010016952 Food poisoning Diseases 0.000 description 2
- 208000019331 Foodborne disease Diseases 0.000 description 2
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerol Natural products OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
- 239000004471 Glycine Substances 0.000 description 2
- 108700000788 Human immunodeficiency virus 1 tat peptide (47-57) Proteins 0.000 description 2
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 2
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 2
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 2
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 2
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 2
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 2
- 229920000168 Microcrystalline cellulose Polymers 0.000 description 2
- 239000002202 Polyethylene glycol Substances 0.000 description 2
- ATUOYWHBWRKTHZ-UHFFFAOYSA-N Propane Chemical compound CCC ATUOYWHBWRKTHZ-UHFFFAOYSA-N 0.000 description 2
- 241000589776 Pseudomonas putida Species 0.000 description 2
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 description 2
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 229920002472 Starch Polymers 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 2
- 206010052428 Wound Diseases 0.000 description 2
- 208000027418 Wounds and injury Diseases 0.000 description 2
- XLOMVQKBTHCTTD-UHFFFAOYSA-N Zinc monoxide Chemical compound [Zn]=O XLOMVQKBTHCTTD-UHFFFAOYSA-N 0.000 description 2
- 206010000269 abscess Diseases 0.000 description 2
- 239000013543 active substance Substances 0.000 description 2
- 235000004279 alanine Nutrition 0.000 description 2
- 235000001014 amino acid Nutrition 0.000 description 2
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 2
- 239000011324 bead Substances 0.000 description 2
- 239000000440 bentonite Substances 0.000 description 2
- 229910000278 bentonite Inorganic materials 0.000 description 2
- SVPXDRXYRYOSEX-UHFFFAOYSA-N bentoquatam Chemical compound O.O=[Si]=O.O=[Al]O[Al]=O SVPXDRXYRYOSEX-UHFFFAOYSA-N 0.000 description 2
- SESFRYSPDFLNCH-UHFFFAOYSA-N benzyl benzoate Chemical compound C=1C=CC=CC=1C(=O)OCC1=CC=CC=C1 SESFRYSPDFLNCH-UHFFFAOYSA-N 0.000 description 2
- 235000013361 beverage Nutrition 0.000 description 2
- 230000032770 biofilm formation Effects 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- 239000000378 calcium silicate Substances 0.000 description 2
- 229910052918 calcium silicate Inorganic materials 0.000 description 2
- 235000012241 calcium silicate Nutrition 0.000 description 2
- OYACROKNLOSFPA-UHFFFAOYSA-N calcium;dioxido(oxo)silane Chemical compound [Ca+2].[O-][Si]([O-])=O OYACROKNLOSFPA-UHFFFAOYSA-N 0.000 description 2
- 239000002775 capsule Substances 0.000 description 2
- 239000001569 carbon dioxide Substances 0.000 description 2
- 229910002092 carbon dioxide Inorganic materials 0.000 description 2
- 239000013592 cell lysate Substances 0.000 description 2
- 239000001913 cellulose Substances 0.000 description 2
- 235000010980 cellulose Nutrition 0.000 description 2
- 229920002678 cellulose Polymers 0.000 description 2
- 235000009508 confectionery Nutrition 0.000 description 2
- 238000012136 culture method Methods 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 238000012217 deletion Methods 0.000 description 2
- 230000037430 deletion Effects 0.000 description 2
- 239000003995 emulsifying agent Substances 0.000 description 2
- 239000000839 emulsion Substances 0.000 description 2
- 206010014665 endocarditis Diseases 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 239000008187 granular material Substances 0.000 description 2
- 235000013402 health food Nutrition 0.000 description 2
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 2
- 230000002163 immunogen Effects 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 2
- 239000008101 lactose Substances 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 235000019813 microcrystalline cellulose Nutrition 0.000 description 2
- 239000008108 microcrystalline cellulose Substances 0.000 description 2
- 229940016286 microcrystalline cellulose Drugs 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 239000003921 oil Substances 0.000 description 2
- 235000019198 oils Nutrition 0.000 description 2
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 2
- 229920001223 polyethylene glycol Polymers 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 239000000377 silicon dioxide Substances 0.000 description 2
- 210000004927 skin cell Anatomy 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 239000000600 sorbitol Substances 0.000 description 2
- 235000010356 sorbitol Nutrition 0.000 description 2
- 239000007921 spray Substances 0.000 description 2
- 239000003381 stabilizer Substances 0.000 description 2
- 239000008107 starch Substances 0.000 description 2
- 235000019698 starch Nutrition 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 239000000375 suspending agent Substances 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 208000011580 syndromic disease Diseases 0.000 description 2
- 239000006188 syrup Substances 0.000 description 2
- 235000020357 syrup Nutrition 0.000 description 2
- 239000003826 tablet Substances 0.000 description 2
- 239000003053 toxin Substances 0.000 description 2
- 231100000765 toxin Toxicity 0.000 description 2
- 108700012359 toxins Proteins 0.000 description 2
- 239000001974 tryptic soy broth Substances 0.000 description 2
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 2
- 239000011782 vitamin Substances 0.000 description 2
- 235000013343 vitamin Nutrition 0.000 description 2
- 229940088594 vitamin Drugs 0.000 description 2
- 229930003231 vitamin Natural products 0.000 description 2
- JNYAEWCLZODPBN-JGWLITMVSA-N (2r,3r,4s)-2-[(1r)-1,2-dihydroxyethyl]oxolane-3,4-diol Chemical compound OC[C@@H](O)[C@H]1OC[C@H](O)[C@H]1O JNYAEWCLZODPBN-JGWLITMVSA-N 0.000 description 1
- LNAZSHAWQACDHT-XIYTZBAFSA-N (2r,3r,4s,5r,6s)-4,5-dimethoxy-2-(methoxymethyl)-3-[(2s,3r,4s,5r,6r)-3,4,5-trimethoxy-6-(methoxymethyl)oxan-2-yl]oxy-6-[(2r,3r,4s,5r,6r)-4,5,6-trimethoxy-2-(methoxymethyl)oxan-3-yl]oxyoxane Chemical compound CO[C@@H]1[C@@H](OC)[C@H](OC)[C@@H](COC)O[C@H]1O[C@H]1[C@H](OC)[C@@H](OC)[C@H](O[C@H]2[C@@H]([C@@H](OC)[C@H](OC)O[C@@H]2COC)OC)O[C@@H]1COC LNAZSHAWQACDHT-XIYTZBAFSA-N 0.000 description 1
- RAVVEEJGALCVIN-AGVBWZICSA-N (2s)-2-[[(2s)-2-[[(2s)-2-[[(2s)-5-amino-2-[[(2s)-2-[[(2s)-2-[[(2s)-6-amino-2-[[(2s)-6-amino-2-[[(2s)-2-[[2-[[(2s)-2-amino-3-(4-hydroxyphenyl)propanoyl]amino]acetyl]amino]-5-(diaminomethylideneamino)pentanoyl]amino]hexanoyl]amino]hexanoyl]amino]-5-(diamino Chemical compound NC(N)=NCCC[C@@H](C(O)=O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCCN=C(N)N)NC(=O)CNC(=O)[C@@H](N)CC1=CC=C(O)C=C1 RAVVEEJGALCVIN-AGVBWZICSA-N 0.000 description 1
- WNWHHMBRJJOGFJ-UHFFFAOYSA-N 16-methylheptadecan-1-ol Chemical class CC(C)CCCCCCCCCCCCCCCO WNWHHMBRJJOGFJ-UHFFFAOYSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- AZKSAVLVSZKNRD-UHFFFAOYSA-M 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide Chemical compound [Br-].S1C(C)=C(C)N=C1[N+]1=NC(C=2C=CC=CC=2)=NN1C1=CC=CC=C1 AZKSAVLVSZKNRD-UHFFFAOYSA-M 0.000 description 1
- OSJPPGNTCRNQQC-UWTATZPHSA-N 3-phospho-D-glyceric acid Chemical compound OC(=O)[C@H](O)COP(O)(O)=O OSJPPGNTCRNQQC-UWTATZPHSA-N 0.000 description 1
- FHVDTGUDJYJELY-UHFFFAOYSA-N 6-{[2-carboxy-4,5-dihydroxy-6-(phosphanyloxy)oxan-3-yl]oxy}-4,5-dihydroxy-3-phosphanyloxane-2-carboxylic acid Chemical compound O1C(C(O)=O)C(P)C(O)C(O)C1OC1C(C(O)=O)OC(OP)C(O)C1O FHVDTGUDJYJELY-UHFFFAOYSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- XUGATJVGQUGQKY-ULQDDVLXSA-N Arg-Lys-Phe Chemical compound NC(=N)NCCC[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 XUGATJVGQUGQKY-ULQDDVLXSA-N 0.000 description 1
- 206010060968 Arthritis infective Diseases 0.000 description 1
- QEYJFBMTSMLPKZ-ZKWXMUAHSA-N Asn-Ala-Val Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](C(C)C)C(O)=O QEYJFBMTSMLPKZ-ZKWXMUAHSA-N 0.000 description 1
- NJIKKGUVGUBICV-ZLUOBGJFSA-N Asp-Ala-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC(O)=O NJIKKGUVGUBICV-ZLUOBGJFSA-N 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 241000194110 Bacillus sp. (in: Bacteria) Species 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- 208000031729 Bacteremia Diseases 0.000 description 1
- 108010006654 Bleomycin Proteins 0.000 description 1
- 101100315624 Caenorhabditis elegans tyr-1 gene Proteins 0.000 description 1
- 206010007882 Cellulitis Diseases 0.000 description 1
- 101710098119 Chaperonin GroEL 2 Proteins 0.000 description 1
- 206010011409 Cross infection Diseases 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 1
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- YQYJSBFKSSDGFO-UHFFFAOYSA-N Epihygromycin Natural products OC1C(O)C(C(=O)C)OC1OC(C(=C1)O)=CC=C1C=C(C)C(=O)NC1C(O)C(O)C2OCOC2C1O YQYJSBFKSSDGFO-UHFFFAOYSA-N 0.000 description 1
- 239000004593 Epoxy Substances 0.000 description 1
- 241000701959 Escherichia virus Lambda Species 0.000 description 1
- 206010015988 Eyelid infection Diseases 0.000 description 1
- 108091092584 GDNA Proteins 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 108700039691 Genetic Promoter Regions Proteins 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- LERGJIVJIIODPZ-ZANVPECISA-N Gly-Ala-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@@H](NC(=O)CN)C)C(O)=O)=CNC2=C1 LERGJIVJIIODPZ-ZANVPECISA-N 0.000 description 1
- LCRDMSSAKLTKBU-ZDLURKLDSA-N Gly-Ser-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)CN LCRDMSSAKLTKBU-ZDLURKLDSA-N 0.000 description 1
- UIQGJYUEQDOODF-KWQFWETISA-N Gly-Tyr-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)CN)CC1=CC=C(O)C=C1 UIQGJYUEQDOODF-KWQFWETISA-N 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- SLQVFYWBGNNOTK-BYULHYEWSA-N Ile-Gly-Asn Chemical compound CC[C@H](C)[C@@H](C(=O)NCC(=O)N[C@@H](CC(=O)N)C(=O)O)N SLQVFYWBGNNOTK-BYULHYEWSA-N 0.000 description 1
- 206010061598 Immunodeficiency Diseases 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 1
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 1
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 1
- IRMLZWSRWSGTOP-CIUDSAMLSA-N Leu-Ser-Ala Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(O)=O IRMLZWSRWSGTOP-CIUDSAMLSA-N 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 239000006137 Luria-Bertani broth Substances 0.000 description 1
- NCZIQZYZPUPMKY-PPCPHDFISA-N Lys-Ile-Thr Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(O)=O NCZIQZYZPUPMKY-PPCPHDFISA-N 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 201000009906 Meningitis Diseases 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 1
- JOCBASBOOFNAJA-UHFFFAOYSA-N N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid Chemical compound OCC(CO)(CO)NCCS(O)(=O)=O JOCBASBOOFNAJA-UHFFFAOYSA-N 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 206010031252 Osteomyelitis Diseases 0.000 description 1
- 101150012394 PHO5 gene Proteins 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 108700019535 Phosphoprotein Phosphatases Proteins 0.000 description 1
- 102000045595 Phosphoprotein Phosphatases Human genes 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 108091000080 Phosphotransferase Proteins 0.000 description 1
- 206010035664 Pneumonia Diseases 0.000 description 1
- 229920003171 Poly (ethylene oxide) Polymers 0.000 description 1
- 239000004952 Polyamide Substances 0.000 description 1
- 206010067268 Post procedural infection Diseases 0.000 description 1
- 241000588770 Proteus mirabilis Species 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- 239000012979 RPMI medium Substances 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 206010040070 Septic Shock Diseases 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- RDFQNDHEHVSONI-ZLUOBGJFSA-N Ser-Asn-Ser Chemical compound OC[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(O)=O RDFQNDHEHVSONI-ZLUOBGJFSA-N 0.000 description 1
- BNFVPSRLHHPQKS-WHFBIAKZSA-N Ser-Asp-Gly Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(O)=O BNFVPSRLHHPQKS-WHFBIAKZSA-N 0.000 description 1
- WLJPJRGQRNCIQS-ZLUOBGJFSA-N Ser-Ser-Asn Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(O)=O WLJPJRGQRNCIQS-ZLUOBGJFSA-N 0.000 description 1
- 206010041925 Staphylococcal infections Diseases 0.000 description 1
- 241000191940 Staphylococcus Species 0.000 description 1
- 101000794214 Staphylococcus aureus Toxic shock syndrome toxin-1 Proteins 0.000 description 1
- 101100509033 Staphylococcus aureus isaA gene Proteins 0.000 description 1
- 241000187747 Streptomyces Species 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- 239000007994 TES buffer Substances 0.000 description 1
- 244000269722 Thea sinensis Species 0.000 description 1
- DJDSEDOKJTZBAR-ZDLURKLDSA-N Thr-Gly-Ser Chemical compound C[C@@H](O)[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O DJDSEDOKJTZBAR-ZDLURKLDSA-N 0.000 description 1
- 206010044248 Toxic shock syndrome Diseases 0.000 description 1
- 231100000650 Toxic shock syndrome Toxicity 0.000 description 1
- OVBMCNDKCWAXMZ-NAKRPEOUSA-N Val-Ile-Ser Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)O)NC(=O)[C@H](C(C)C)N OVBMCNDKCWAXMZ-NAKRPEOUSA-N 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 238000007792 addition Methods 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 238000004115 adherent culture Methods 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 108010070783 alanyltyrosine Proteins 0.000 description 1
- 235000013334 alcoholic beverage Nutrition 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 229940072056 alginate Drugs 0.000 description 1
- 235000010443 alginic acid Nutrition 0.000 description 1
- 229920000615 alginic acid Polymers 0.000 description 1
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 1
- 229910052782 aluminium Inorganic materials 0.000 description 1
- WNROFYMDJYEPJX-UHFFFAOYSA-K aluminium hydroxide Chemical compound [OH-].[OH-].[OH-].[Al+3] WNROFYMDJYEPJX-UHFFFAOYSA-K 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 239000010775 animal oil Substances 0.000 description 1
- 230000000845 anti-microbial effect Effects 0.000 description 1
- 230000000941 anti-staphylcoccal effect Effects 0.000 description 1
- 229940124350 antibacterial drug Drugs 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 239000012736 aqueous medium Substances 0.000 description 1
- 108010068265 aspartyltyrosine Proteins 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 229960002903 benzyl benzoate Drugs 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 229960001561 bleomycin Drugs 0.000 description 1
- OYVAGSVQBOHSSS-UAPAGMARSA-O bleomycin A2 Chemical compound N([C@H](C(=O)N[C@H](C)[C@@H](O)[C@H](C)C(=O)N[C@@H]([C@H](O)C)C(=O)NCCC=1SC=C(N=1)C=1SC=C(N=1)C(=O)NCCC[S+](C)C)[C@@H](O[C@H]1[C@H]([C@@H](O)[C@H](O)[C@H](CO)O1)O[C@@H]1[C@H]([C@@H](OC(N)=O)[C@H](O)[C@@H](CO)O1)O)C=1N=CNC=1)C(=O)C1=NC([C@H](CC(N)=O)NC[C@H](N)C(N)=O)=NC(N)=C1C OYVAGSVQBOHSSS-UAPAGMARSA-O 0.000 description 1
- 235000008429 bread Nutrition 0.000 description 1
- 239000001273 butane Substances 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 229960001714 calcium phosphate Drugs 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 229960003340 calcium silicate Drugs 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 229960005091 chloramphenicol Drugs 0.000 description 1
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 1
- 150000005827 chlorofluoro hydrocarbons Chemical class 0.000 description 1
- 235000019219 chocolate Nutrition 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000010411 cooking Methods 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 231100000263 cytotoxicity test Toxicity 0.000 description 1
- 235000013365 dairy product Nutrition 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 239000008121 dextrose Substances 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 239000002270 dispersing agent Substances 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 230000003511 endothelial effect Effects 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 239000000686 essence Substances 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 230000029142 excretion Effects 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 210000003608 fece Anatomy 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 239000006260 foam Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 235000013373 food additive Nutrition 0.000 description 1
- 239000002778 food additive Substances 0.000 description 1
- 235000013355 food flavoring agent Nutrition 0.000 description 1
- 235000003599 food sweetener Nutrition 0.000 description 1
- 239000003205 fragrance Substances 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 229940014259 gelatin Drugs 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 230000002414 glycolytic effect Effects 0.000 description 1
- 108010048994 glycyl-tyrosyl-alanine Proteins 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 239000008269 hand cream Substances 0.000 description 1
- 235000015243 ice cream Nutrition 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 230000016784 immunoglobulin production Effects 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 238000010255 intramuscular injection Methods 0.000 description 1
- 239000007927 intramuscular injection Substances 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- 239000012139 lysis buffer Substances 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- 229920000609 methyl cellulose Polymers 0.000 description 1
- 239000001923 methylcellulose Substances 0.000 description 1
- LXCFILQKKLGQFO-UHFFFAOYSA-N methylparaben Chemical compound COC(=O)C1=CC=C(O)C=C1 LXCFILQKKLGQFO-UHFFFAOYSA-N 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 239000002480 mineral oil Substances 0.000 description 1
- 235000010446 mineral oil Nutrition 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- CQDGTJPVBWZJAZ-UHFFFAOYSA-N monoethyl carbonate Chemical compound CCOC(O)=O CQDGTJPVBWZJAZ-UHFFFAOYSA-N 0.000 description 1
- IJDNQMDRQITEOD-UHFFFAOYSA-N n-butane Chemical compound CCCC IJDNQMDRQITEOD-UHFFFAOYSA-N 0.000 description 1
- OFBQJSOFQDEBGM-UHFFFAOYSA-N n-pentane Natural products CCCCC OFBQJSOFQDEBGM-UHFFFAOYSA-N 0.000 description 1
- 235000012149 noodles Nutrition 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 239000002674 ointment Substances 0.000 description 1
- 239000011022 opal Substances 0.000 description 1
- 235000019449 other food additives Nutrition 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 238000007911 parenteral administration Methods 0.000 description 1
- 239000006072 paste Substances 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 238000013148 permeation assay Methods 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- WVDDGKGOMKODPV-ZQBYOMGUSA-N phenyl(114C)methanol Chemical compound O[14CH2]C1=CC=CC=C1 WVDDGKGOMKODPV-ZQBYOMGUSA-N 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 102000020233 phosphotransferase Human genes 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 239000006187 pill Substances 0.000 description 1
- 235000013550 pizza Nutrition 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 229920002647 polyamide Polymers 0.000 description 1
- 229920001296 polysiloxane Polymers 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 210000001236 prokaryotic cell Anatomy 0.000 description 1
- 239000001294 propane Substances 0.000 description 1
- 239000003380 propellant Substances 0.000 description 1
- 230000000069 prophylactic effect Effects 0.000 description 1
- QELSKZZBTMNZEB-UHFFFAOYSA-N propylparaben Chemical compound CCCOC(=O)C1=CC=C(O)C=C1 QELSKZZBTMNZEB-UHFFFAOYSA-N 0.000 description 1
- 229960003415 propylparaben Drugs 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- BOLDJAUMGUJJKM-LSDHHAIUSA-N renifolin D Natural products CC(=C)[C@@H]1Cc2c(O)c(O)ccc2[C@H]1CC(=O)c3ccc(O)cc3O BOLDJAUMGUJJKM-LSDHHAIUSA-N 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 235000013580 sausages Nutrition 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 239000002453 shampoo Substances 0.000 description 1
- 206010040872 skin infection Diseases 0.000 description 1
- 235000011888 snacks Nutrition 0.000 description 1
- 239000000344 soap Substances 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 235000014347 soups Nutrition 0.000 description 1
- 208000015339 staphylococcus aureus infection Diseases 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 230000000475 sunscreen effect Effects 0.000 description 1
- 239000000516 sunscreening agent Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000000829 suppository Substances 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 238000004114 suspension culture Methods 0.000 description 1
- 239000003765 sweetening agent Substances 0.000 description 1
- 235000013616 tea Nutrition 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 230000001256 tonic effect Effects 0.000 description 1
- 238000011200 topical administration Methods 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 108010050327 trypticase-soy broth Proteins 0.000 description 1
- 241000701161 unidentified adenovirus Species 0.000 description 1
- 241001148471 unidentified anaerobic bacterium Species 0.000 description 1
- 241001430294 unidentified retrovirus Species 0.000 description 1
- 235000015112 vegetable and seed oil Nutrition 0.000 description 1
- 239000008158 vegetable oil Substances 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
- 239000001993 wax Substances 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
- 239000011787 zinc oxide Substances 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/56938—Staphylococcus
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2200/00—Function of food ingredients
- A23V2200/30—Foods, ingredients or supplements having a functional effect on health
- A23V2200/318—Foods, ingredients or supplements having a functional effect on health having an effect on skin health and hair or coat
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/924—Hydrolases (3) acting on glycosyl compounds (3.2)
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Organic Chemistry (AREA)
- Medicinal Chemistry (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Biochemistry (AREA)
- Hematology (AREA)
- Dermatology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Food Science & Technology (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Analytical Chemistry (AREA)
- Biophysics (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- General Chemical & Material Sciences (AREA)
- Physics & Mathematics (AREA)
- Pharmacology & Pharmacy (AREA)
- Virology (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Tropical Medicine & Parasitology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Birds (AREA)
- Epidemiology (AREA)
- Mycology (AREA)
- Nutrition Science (AREA)
- Polymers & Plastics (AREA)
Abstract
본 발명은 황색포도상구균에 특이적으로 반응하는 항-IsaA G4 항체 및 이의 용도에 관한 것으로서, 더욱 상세하게, 본 발명은 특정 서열의 중쇄 CDR 및 경쇄 CDR을 포함하는 항-IsaA G4 항체 또는 이의 항원 결합 단편에 대한 것이다. 상기 항-IsaA G4 항체는 황색포도상구균의 검출에 활용될 뿐만 아니라, 황색포도상구균 성장을 억제하여 아토피 질환과 같은 피부 염증 개선 또는 치료에 유용하게 활용될 수 있을 것으로 예상된다.The present invention relates to an anti-IsaA G4 antibody specifically responding to Staphylococcus aureus and a use thereof, and more particularly, the present invention relates to an anti-IsaA G4 antibody or antigen thereof comprising heavy and light chain CDRs of specific sequences to the binding fragment. The anti-IsaA G4 antibody is expected to be useful not only for the detection of Staphylococcus aureus, but also for the improvement or treatment of skin inflammation such as atopic diseases by inhibiting the growth of Staphylococcus aureus.
Description
본 발명은 황색포도상구균에 특이적으로 반응하는 항-IsaA G4 항체 및 이의 용도에 대한 것이다. The present invention relates to anti-IsaA G4 antibodies specifically reactive against Staphylococcus aureus and uses thereof.
황색포도상구균(Staphylococcus aureus)은 그람 양성균이며, 포자를 형성하지 않는 통성혐기성균으로, 주로 가축 분변, 염증 상처부위 혹은 사람의 손이 많이 닿는 조리도구 등에서 발견된다. 인간과 동물에 감염하여 수술 후 감염, 농양, 심장 내막염 및 독소 증후군 등의 심각한 질병을 유도하는 대표적인 병원성 세균이다. 황색포도상구균이 내는 TSST-1, EF, alpha, beta, delta 등의 독소는 심각한 식중독을 유발하기도 한다. 또한, 황색포도상구균 감염은 다양한 질환 또는 병태(condition)를 야기할 수 있으며, 이들 질환 또는 병태의 비-한정적 예로는 세균혈증, 연조직염, 눈꺼풀 감염, 식중독, 관절 감염, 피부 감염, 열상 피부 증후군, 독성 쇼크 증후군, 폐렴, 골수염, 심내막염, 수막염 및 농양 형성이 있다.Staphylococcus aureus is a Gram-positive, non-spore-forming, facultative anaerobic bacteria, mainly found in livestock feces, inflamed wounds, or cooking utensils that are touched by people's hands. It is a representative pathogenic bacterium that infects humans and animals and induces serious diseases such as postoperative infections, abscesses, endocarditis, and toxin syndrome. Toxins such as TSST-1, EF, alpha, beta, and delta produced by Staphylococcus aureus can cause serious food poisoning. In addition, Staphylococcus aureus infection can cause a variety of diseases or conditions, non-limiting examples of which include bacteremia, cellulitis, eyelid infection, food poisoning, joint infection, skin infection, lacerated skin syndrome. , toxic shock syndrome, pneumonia, osteomyelitis, endocarditis, meningitis and abscess formation.
한편, 황색포도상구균은 병원내 감염을 일으키는 대표적인 세균으로 정상인이나 환경에서도 쉽게 발견되는 상재세균이다. 이 세균은 상처나 면역저하환자, 그리고 외과적 치료부위에 오염된 의료기구 등을 통하여 감염하여 항균제로 치료하기 어려운 내성을 가지고 화농성 질환을 일으킨다. 균의 특성상 의료기구나 비생물체의 표면에 고착하여 오랫동안 생존하고 있기 때문에 일반적인 세제나 알코올류로 완전히 제거가 어렵다. 이를 세균의 균막형성이라 하며 균막형성시 치료가 더욱 어렵고 항균제에도 보다 높은 저항성을 나타낸다. 인체 감염시 균막은 인체의 면역체계에 저항하며, 항균물질에 대한 저항성이 부유 상태의 500배에서 1000배 이상 증가하는 것으로 나타나 항균치료만으로는 효과적인 치료를 기대하기 힘들다. 뿐만 아니라 항균제 내성균의 출현 및 확산 문제가 매우 심각해서 현재까지 개발되어 사용되고 있는 거의 모든 종류의 항균치료제에 내성을 나타내는 슈퍼박테리아까지 출현하여, 항균제 이외의 대체치료제의 개발이 시급하고 절실하게 요구되고 있다. 그럼에도 불구하고, 황색포도상구균을 억제하는 항체에 대한 연구는 여전히 부족한 실정이다.On the other hand, Staphylococcus aureus is a representative bacterium that causes nosocomial infections and is a resident bacterium that is easily found in normal people and the environment. This bacterium infects wounds, immunocompromised patients, and medical instruments contaminated with surgical treatment sites, and causes purulent diseases with resistance that is difficult to treat with antibacterial agents. Due to the nature of bacteria, they are adhered to the surface of medical devices or non-living objects and survive for a long time, so it is difficult to completely remove them with general detergents or alcohols. This is called bacterial biofilm formation, and it is more difficult to treat during biofilm formation and exhibits higher resistance to antibacterial agents. In the case of human infection, the biofilm resists the body's immune system, and the resistance to antibacterial substances is increased from 500 to 1000 times that of floating state, so it is difficult to expect effective treatment with antibacterial treatment alone. In addition, the emergence and spread of antimicrobial-resistant bacteria is very serious, and even super bacteria that are resistant to almost all types of antibacterial drugs that have been developed and used up to now have appeared. . Nevertheless, studies on antibodies inhibiting Staphylococcus aureus are still lacking.
본 발명의 목적은 황색포도상구균에 특이적으로 반응하는 항-IsaA G4 항체 또는 그의 항원 결합 단편을 제공하는 데에 있다.It is an object of the present invention to provide an anti-IsaA G4 antibody or antigen-binding fragment thereof that specifically reacts against Staphylococcus aureus.
본 발명의 다른 목적은 상기 항-IsaA G4 항체 또는 그의 항원 결합 단편에 추가적으로 TAT 펩타이드가 결합된 융합 항-IsaA G4 항체 또는 그의 항원 결합 단편을 제공하는 데에 있다.Another object of the present invention is to provide a fusion anti-IsaA G4 antibody or antigen-binding fragment thereof in which a TAT peptide is additionally bound to the anti-IsaA G4 antibody or antigen-binding fragment thereof.
본 발명의 또 다른 목적은 상기 항체 또는 그의 항원 결합 단편을 코딩하는 핵산 분자, 상기 핵산 분자를 포함하는 재조합 발현벡터 및 상기 재조합 발현벡터로 형질전환된 세포를 제공하는 데에 있다. Another object of the present invention is to provide a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof, a recombinant expression vector containing the nucleic acid molecule, and cells transformed with the recombinant expression vector.
본 발명의 또 다른 목적은 상기 항체 또는 그의 항원 결합 단편을 유효성분으로 포함하는 IsaA 항원 검출용 조성물을 제공하는 데에 있다. Another object of the present invention is to provide a composition for detecting IsaA antigen comprising the antibody or antigen-binding fragment thereof as an active ingredient.
본 발명의 또 다른 목적은 상기 항체 또는 그의 항원 결합 단편을 유효성분으로 포함하는 황색포도상구균(Staphylococcus aureus) 검출용 조성물을 제공하는 데에 있다. Another object of the present invention is to provide a composition for detecting Staphylococcus aureus comprising the antibody or antigen-binding fragment thereof as an active ingredient.
본 발명의 또 다른 목적은 상기 항체 또는 그의 항원 결합 단편을 유효성분으로 포함하는 황색포도상구균(Staphylococcus aureus) 억제용 항균 조성물을 제공하는 데에 있다. Another object of the present invention is to provide an antibacterial composition for inhibiting Staphylococcus aureus comprising the antibody or antigen-binding fragment thereof as an active ingredient.
본 발명의 또 다른 목적은 상기 항체 또는 그의 항원 결합 단편을 유효성분으로 포함하는 아토피 피부염 예방 또는 개선용 화장료 조성물, 아토피 피부염 예방 또는 개선용 건강기능식품 조성물과, 아토피 피부염 예방 또는 치료용 약학 조성물을 제공하는 데에 있다.Another object of the present invention is a cosmetic composition for preventing or improving atopic dermatitis comprising the antibody or antigen-binding fragment thereof as an active ingredient, a health functional food composition for preventing or improving atopic dermatitis, and a pharmaceutical composition for preventing or treating atopic dermatitis is to provide
상기 목적을 달성하기 위하여, 본 발명은 서열번호 1로 표시되는 아미노산 서열로 이루어진 경쇄 CDR1, 서열번호 2로 표시되는 아미노산 서열로 이루어진 경쇄 CDR2 및 서열번호 3으로 표시되는 아미노산 서열로 이루어진 경쇄 CDR3을 포함하는 경쇄 가변 영역; 및 서열번호 4로 표시되는 아미노산 서열로 이루어진 중쇄 CDR1, 서열번호 5로 표시되는 아미노산 서열로 이루어진 중쇄 CDR2 및 서열번호 6으로 표시되는 아미노산 서열로 이루어진 중쇄 CDR3을 포함하는 중쇄 가변 영역을 포함하는 항-IsaA G4 항체 또는 그의 항원 결합 단편을 제공한다.In order to achieve the above object, the present invention includes a light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 1, a light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 2, and a light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 3 a light chain variable region; and a heavy chain variable region comprising a heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 4, a heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 5, and a heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 6 anti- An IsaA G4 antibody or antigen-binding fragment thereof is provided.
또한, 본 발명은 항체 또는 그의 항원 결합 단편에 추가적으로 서열번호 7로 표시되는 TAT 펩타이드가 결합된 융합 항-IsaA G4 항체 또는 그의 항원 결합 단편을 제공한다.In addition, the present invention provides a fusion anti-IsaA G4 antibody or antigen-binding fragment thereof in which the TAT peptide shown in SEQ ID NO: 7 is additionally bound to the antibody or antigen-binding fragment thereof.
또한, 본 발명은 상기 항체 또는 그의 항원 결합 단편을 코딩하는 핵산 분자를 제공한다.The present invention also provides a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof.
또한, 본 발명은 상기 핵산 분자를 포함하는 재조합 발현벡터를 제공한다.In addition, the present invention provides a recombinant expression vector comprising the nucleic acid molecule.
또한, 본 발명은 상기 재조합 발현벡터로 형질전환된 세포를 제공한다.In addition, the present invention provides a cell transformed with the recombinant expression vector.
또한, 본 발명은 상기 항체 또는 그의 항원 결합 단편을 유효성분으로 포함하는 IsaA 항원 검출용 조성물을 제공한다.In addition, the present invention provides a composition for detecting IsaA antigen comprising the antibody or antigen-binding fragment thereof as an active ingredient.
또한, 본 발명은 상기 항체 또는 그의 항원 결합 단편을 유효성분으로 포함하는 황색포도상구균(Staphylococcus aureus) 검출용 조성물을 제공한다.In addition, the present invention provides a composition for detecting Staphylococcus aureus comprising the antibody or antigen-binding fragment thereof as an active ingredient.
또한, 본 발명은 상기 항체 또는 그의 항원 결합 단편을 유효성분으로 포함하는 황색포도상구균(Staphylococcus aureus) 억제용 항균 조성물을 제공한다.In addition, the present invention provides an antibacterial composition for inhibiting Staphylococcus aureus comprising the antibody or antigen-binding fragment thereof as an active ingredient.
또한, 본 발명은 상기 항체 또는 그의 항원 결합 단편을 유효성분으로 포함하는 아토피 피부염 예방 또는 개선용 화장료 조성물을 제공한다.In addition, the present invention provides a cosmetic composition for preventing or improving atopic dermatitis comprising the antibody or antigen-binding fragment thereof as an active ingredient.
또한, 본 발명은 상기 항체 또는 그의 항원 결합 단편을 유효성분으로 포함하는 아토피 피부염 예방 또는 개선용 건강기능식품 조성물을 제공한다.In addition, the present invention provides a health functional food composition for preventing or improving atopic dermatitis comprising the antibody or antigen-binding fragment thereof as an active ingredient.
또한, 본 발명은 상기 항체 또는 그의 항원 결합 단편을 유효성분으로 포함하는 아토피 피부염 예방 또는 치료용 약학조성물을 제공한다.In addition, the present invention provides a pharmaceutical composition for preventing or treating atopic dermatitis comprising the antibody or antigen-binding fragment thereof as an active ingredient.
본 발명은 황색포도상구균에 특이적으로 반응하는 항-IsaA G4 항체 및 이의 용도에 관한 것으로서, 더욱 상세하게, 본 발명은 특정 서열의 중쇄 CDR 및 경쇄 CDR을 포함하는 항-IsaA G4 항체 또는 이의 항원 결합 단편에 대한 것이다. 상기 항-IsaA G4 항체는 황색포도상구균의 검출에 활용될 뿐만 아니라, 황색포도상구균 성장을 억제하여 아토피 질환과 같은 피부 염증 개선 또는 치료에 유용하게 활용될 수 있을 것으로 예상된다.The present invention relates to an anti-IsaA G4 antibody specifically responding to Staphylococcus aureus and a use thereof, and more particularly, the present invention relates to an anti-IsaA G4 antibody or antigen thereof comprising heavy and light chain CDRs of specific sequences to the binding fragment. The anti-IsaA G4 antibody is expected to be useful not only for the detection of Staphylococcus aureus, but also for the improvement or treatment of skin inflammation such as atopic diseases by inhibiting the growth of Staphylococcus aureus.
도 1은 항-IsaA scFv 항체 선별을 위한 ELISA 분석 결과를 나타낸 것이다.
도 2는 항-IsaA (G4) scFv 항체의 민감도 분석 결과를 나타낸 것이다.
도 3은 황색포도상구균을 이용한 항-IsaA (G4) scFv 항체의 활성도 분석 결과를 나타낸 것이다.
도 4은 세포투과성 항-IsaA (G4) scFv 항체 단백질 정제 후 SDS-PAGE 분석법을 통해 크기 및 단백질 순도 결과를 나타낸 것이다.
도 5는 세포투과성 항-IsaA (G4) scFv 항체 단백질의 세포내 투과능을 웨스턴 블랏 분석법으로 결과를 나타낸 것이다.1 shows the results of ELISA analysis for anti-IsaA scFv antibody selection.
Figure 2 shows the results of the sensitivity analysis of the anti-IsaA (G4) scFv antibody.
Figure 3 shows the results of the activity analysis of the anti-IsaA (G4) scFv antibody using Staphylococcus aureus.
Figure 4 shows the size and protein purity results through SDS-PAGE analysis after cell-permeable anti-IsaA (G4) scFv antibody protein purification.
FIG. 5 shows the results of intracellular permeability of the cell-permeable anti-IsaA (G4) scFv antibody protein by Western blot analysis.
본 발명은 서열번호 1로 표시되는 아미노산 서열로 이루어진 경쇄 CDR1, 서열번호 2로 표시되는 아미노산 서열로 이루어진 경쇄 CDR2 및 서열번호 3으로 표시되는 아미노산 서열로 이루어진 경쇄 CDR3을 포함하는 경쇄 가변 영역; 및 서열번호 4로 표시되는 아미노산 서열로 이루어진 중쇄 CDR1, 서열번호 5로 표시되는 아미노산 서열로 이루어진 중쇄 CDR2 및 서열번호 6으로 표시되는 아미노산 서열로 이루어진 중쇄 CDR3을 포함하는 중쇄 가변 영역을 포함하는 항-IsaA G4 항체 또는 그의 항원 결합 단편을 제공한다.The present invention provides a light chain variable region comprising a light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 1, a light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 2, and a light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 3; and a heavy chain variable region comprising a heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 4, a heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 5, and a heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 6 anti- An IsaA G4 antibody or antigen-binding fragment thereof is provided.
또한, 본 발명은 항체 또는 그의 항원 결합 단편에 추가적으로 서열번호 7로 표시되는 TAT 펩타이드가 결합된 융합 항-IsaA G4 항체 또는 그의 항원 결합 단편을 제공한다.In addition, the present invention provides a fusion anti-IsaA G4 antibody or antigen-binding fragment thereof in which the TAT peptide shown in SEQ ID NO: 7 is additionally bound to the antibody or antigen-binding fragment thereof.
한편, 서열번호 1 내지 서열번호 6로 표시되는 아미노산으로 이루어진 CDR들은 표 1에 기재하였다.On the other hand, the CDRs consisting of amino acids represented by SEQ ID NO: 1 to SEQ ID NO: 6 are described in Table 1.
또한, 본 발명에 사용된 TAT 펩타이드의 아미노산 서열은 "YGRKKRRQRRR" (서열번호 7)이고, TAT 펩타이드의 염기서열은 "TAT GGC CGC AAA AAA CGC CGC CAG CGC CGC CGC" (서열번호 8)이다. In addition, the amino acid sequence of the TAT peptide used in the present invention is "YGRKKRRQRRR" (SEQ ID NO: 7), and the nucleotide sequence of the TAT peptide is "TAT GGC CGC AAA AAA CGC CGC CAG CGC CGC CGC" (SEQ ID NO: 8).
본 발명에서 용어, “항체”는 면역학적으로 특정 항원과 반응성을 갖는 면역글로불린 분자를 포함하는, 항원을 특이적으로 인식하는 수용체 역할을 하는 단백질 분자를 의미하며, 그 예로, 단일 클론 항체, 다클론 항체, 전장항체(full-length antibody) 및 항체 단편을 모두 포함할 수 있다. 또한 상기 용어, “항체”는 이가(bivalent) 또는 이중 특이성 분자(예컨대, 이중특이성 항체), 디아바디, 트리아바디 또는 테트라바디를 포함할 수 있다.As used herein, the term “antibody” refers to a protein molecule serving as a receptor that specifically recognizes an antigen, including an immunoglobulin molecule that is immunologically reactive with a specific antigen, for example, a monoclonal antibody, It may include all of a clonal antibody, a full-length antibody, and an antibody fragment. Also, the term “antibody” may include a bivalent or bispecific molecule (eg, a bispecific antibody), a diabody, a triabody, or a tetrabody.
본 발명에서 용어, “단일 클론 항체”는 실질적으로 동일한 항체 집단에서 수득한 단일 분자 조성의 항체 분자를 지칭하고, 이러한 단일 클론 항체는 다클론 항체가 여러 개의 에피토프에 결합할 수 있는 것과 달리, 특정 에피토프에 대해 단일 결합성 및 친화도를 나타낸다. 본 발명에서 용어, “전장항체”는 2 개의 전체 길이의 경쇄 및 2 개의 전체 길이의 중쇄를 가지는 구조이며, 각각의 경쇄는 중쇄와 다이설파이드 결합으로 연결되어 있다. 중쇄 불변영역은 감마(γ), 뮤(μ), 알파(α), 델타(δ) 및 엡실론(ε) 타입을 가지고 서브클래스로 감마1(γ1), 감마2(γ2), 감마3(γ3), 감마4(γ4), 알파1(α1) 및 알파2(α2)를 가진다. 경쇄의 불변영역은 카파(κ) 및 람다(λ) 타입을 가진다. IgG는 서브타입(subtype)으로, IgG1, IgG2, IgG3 및 IgG4를 포함한다.As used herein, the term "monoclonal antibody" refers to an antibody molecule having a single molecular composition obtained from a population of substantially identical antibodies, and such monoclonal antibody is different from a polyclonal antibody that can bind to multiple epitopes, It exhibits single binding and affinity for the epitope. As used herein, the term “full-length antibody” has a structure having two full-length light chains and two full-length heavy chains, and each light chain is connected to the heavy chain by a disulfide bond. The heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ) and epsilon (ε) types and subclasses gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3). ), gamma 4 (γ4), alpha 1 (α1) and alpha 2 (α2). The constant region of the light chain has a kappa (κ) and a lambda (λ) type. IgG is a subtype and includes IgG1, IgG2, IgG3 and IgG4.
본 발명에서 용어, “중쇄”는 항원에 특이성을 부여하기 위한 충분한 가변영역 서열을 갖는 아미노산 서열을 포함하는 가변 영역 VH 및 3 개의 불변 영역 CH1, CH2 및 CH3를 포함하는 전체길이 중쇄 및 이의 단편을 모두 포함할 수 있다. 또한, 본 발명에서 용어, “경쇄”는 항원에 특이성을 부여하기 위한 충분한 가변영역 서열을 갖는 아미노산 서열을 포함하는 가변 영역 VL 및 불변 영역 CL을 포함하는 전체길이 경쇄 및 이의 단편을 모두 포함할 수 있다.As used herein, the term “heavy chain” refers to a full-length heavy chain comprising a variable region VH comprising an amino acid sequence having a sufficient variable region sequence to confer specificity to an antigen and three constant regions CH1, CH2 and CH3, and fragments thereof. can include all of them. In addition, as used herein, the term “light chain” may include both a full-length light chain including a variable region VL and a constant region CL comprising an amino acid sequence having a sufficient variable region sequence to confer specificity to an antigen, and fragments thereof. have.
본 발명에서 용어, “단편”, “항체 단편” 및 “항원 결합 단편”은 항체의 항원결합 기능을 보유하는 본 발명의 항체의 임의의 단편을 지칭하는 것으로 호환적으로 사용된다. 예시적인 항원 결합 단편은 Fab, Fab', F(ab')2 및 Fv 등을 포함하나, 이에 제한되지 않는다.In the present invention, the terms “fragment”, “antibody fragment” and “antigen-binding fragment” are used interchangeably to refer to any fragment of an antibody of the present invention that retains the antigen-binding function of the antibody. Exemplary antigen binding fragments include, but are not limited to, Fab, Fab', F(ab')2 and Fv, and the like.
본 발명의 항체 또는 그의 항원 결합 단편은 IsaA에 특이적으로 결합하는 능력을 나타낼 수 있는 범위 내에서, 본 명세서에 기재된 항체의 서열뿐만 아니라, 이의 생물학적 균등물도 포함할 수 있다. 예를 들면, 항체의 결합 친화도 및/또는 기타 생물학적 특성을 보다 더 개선시키기 위하여 항체의 아미노산 서열에 추가적인 변화를 줄 수 있다. 이러한 변형은 예를 들어, 항체의 아미노산 서열 잔기의 결실, 삽입 및/또는 치환을 포함한다. 이러한 아미노산 변이는 아미노산 곁사슬 치환체의 상대적 유사성, 예컨대, 소수성, 친수성, 전하, 크기 등에 기초하여 이루어진다. 아미노산 곁사슬 치환체의 크기, 모양 및 종류에 대한 분석에 의하여, 아르기닌, 리신과 히스티딘은 모두 양전하를 띈 잔기이고; 알라닌, 글리신과 세린은 유사한 크기를 가지며; 페닐알라닌, 트립토판과 티로신은 유사한 모양을 갖는다는 것을 알 수 있다. 따라서, 이점에 기초하여, 아르기닌, 라신과 히스티딘; 알라닌, 글리신과 세린; 그리고 페닐알라닌, 트립토판과 티로신은 생물학적으로 기능 균등물이라 할 수 있다.The antibody or antigen-binding fragment thereof of the present invention may include not only the sequence of the antibody described herein, but also a biological equivalent thereof to the extent that it can exhibit the ability to specifically bind to IsaA. For example, additional changes may be made to the amino acid sequence of an antibody to further improve its binding affinity and/or other biological properties. Such modifications include, for example, deletions, insertions and/or substitutions of amino acid sequence residues of the antibody. Such amino acid variations are made based on the relative similarity of amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, size, and the like. According to analysis of the size, shape and type of amino acid side chain substituents, arginine, lysine and histidine are all positively charged residues; Alanine, glycine and serine have similar sizes; It can be seen that phenylalanine, tryptophan and tyrosine have similar shapes. Therefore, on the basis of this, arginine, racine and histidine; alanine, glycine and serine; And phenylalanine, tryptophan and tyrosine can be said to be biologically functional equivalents.
또한, 본 발명은 상기 항체 또는 그의 항원 결합 단편을 코딩하는 핵산 분자를 제공한다.The present invention also provides a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof.
본 명세서에서 사용되는 용어, “핵산 분자”는 DNA(gDNA 및 cDNA) 및 RNA 분자를 포괄적으로 포함하는 의미를 가지며, 핵산 분자에서 기본 구성단위인 뉴클레오티드는 자연의 뉴클레오티드뿐만 아니라, 당 또는 염기 부위가 변형된 유사체(analogue)도 포함한다. 본 발명의 중쇄 및 경쇄 가변 영역을 코딩하는 핵산 분자의 서열은 변형될 수 있으며, 상기 변형은 뉴클레오티드의 추가, 결실, 또는 비보존적 치환 또는 보존적 치환을 포함한다.As used herein, the term “nucleic acid molecule” has a meaning comprehensively including DNA (gDNA and cDNA) and RNA molecules. Modified analogs are also included. The sequences of the nucleic acid molecules encoding the heavy and light chain variable regions of the invention may be modified, including additions, deletions, or non-conservative or conservative substitutions of nucleotides.
또한, 본 발명은 상기 핵산 분자를 포함하는 재조합 발현벡터를 제공한다.In addition, the present invention provides a recombinant expression vector comprising the nucleic acid molecule.
본 발명에 있어서, "벡터"는 클론유전자(또는 클론 DNA의 다른 조각)를 운반하는데 사용되는 스스로 복제되는 DNA 분자를 의미한다.As used herein, "vector" refers to a self-replicating DNA molecule used to carry a clonal gene (or another piece of clonal DNA).
본 발명에서 있어서, “발현 벡터”는 목적한 코딩 서열과, 특정 숙주 생물에서 작동 가능하게 연결된 코딩 서열을 발현하는데 필수적인 적정 핵산 서열을 포함하는 재조합 DNA 분자를 의미한다. 발현 벡터는 바람직하게는 하나 이상의 선택성 마커를 포함할 수 있다. 상기 마커는 통상적으로 화학적인 방법으로 선택될 수 있는 특성을 갖는 핵산 서열로, 형질 전환된 세포를 비 형질전환 세포로부터 구별할 수 있는 모든 유전자가 이에 해당된다. 그 예로는 앰피실린(Ampicillin), 카나마이신(Kanamycin), 제네티신(Geneticin; G418), 블레오마이신(Bleomycin), 하이그로마이신(Hygromycin), 클로람페니콜(Chloramphenicol)과 같은 항생제 내성 유전자가 있으나, 이에 한정되는 것은 아니며, 당업자에 의해 적절히 선택 가능하다.In the present invention, "expression vector" refers to a recombinant DNA molecule comprising a desired coding sequence and an appropriate nucleic acid sequence essential for expressing a coding sequence operably linked in a specific host organism. The expression vector may preferably comprise one or more selectable markers. The marker is a nucleic acid sequence having characteristics that can be selected by conventional chemical methods, and includes all genes that can distinguish transformed cells from non-transformed cells. Examples include antibiotic resistance genes such as ampicillin, kanamycin, geneticin (G418), bleomycin, hygromycin, chloramphenicol, but are limited thereto It is not necessary and can be appropriately selected by those skilled in the art.
본 발명의 DNA 서열을 발현시키기 위하여, 매우 다양한 발현 조절 서열 중 어느 것이라도 벡터에 사용될 수 있다. 유용한 발현 조절서열의 예에는, 예를 들어, SV40 또는 아데노바이러스의 초기 및 후기 프로모터들, CMV의 프로모터와 인핸서, 레트로바이러스의 LTR, lac 시스템, trp 시스템, TAC 또는 TRC 시스템, T3 및 T7 프로모터들, 파지 람다의 주요 오퍼레이터 및 프로모터 영역, fd 코드 단백질의 조절 영역, 3-포스포글리세레이트 키나제 또는 다른 글리콜분해 효소에 대한 프로모터, 상기 포스파타제의 프로모터들, 예를 들어 Pho5, 효모 알파-교배 시스템의 프로모터 및 원핵세포 또는 진핵 세포 또는 이들의 바이러스의 유전자의 발현을 조절하는 것으로 알려진 구성과 유도의 기타 다른 서열 및 이들의 여러 조합이 포함될 수 있다.To express the DNA sequences of the present invention, any of a wide variety of expression control sequences can be used in the vector. Examples of useful expression control sequences include, for example, early and late promoters of SV40 or adenovirus, promoters and enhancers of CMV, LTR of retrovirus, lac system, trp system, TAC or TRC system, T3 and T7 promoters , major operator and promoter regions of phage lambda, regulatory regions of fd-coding proteins, promoters for 3-phosphoglycerate kinase or other glycolytic enzymes, promoters of said phosphatases, such as Pho5, yeast alpha-crossing system Promoters and other sequences of construction and induction known to regulate expression of genes in prokaryotic or eukaryotic cells or viruses thereof, and various combinations thereof, may be included.
본 발명의 항체를 발현하는 벡터는, 경쇄와 중쇄가 하나의 벡터에서 동시에 발현되는 벡터 시스템이거나 또는 경쇄와 중쇄를 각각 별도의 벡터에서 발현시키는 시스템 모두 가능하다. 후자의 경우, 두 벡터는 동시 형질전환(co-transfomation) 및 표적 형질전환(targeted transformation)을 통하여 숙주세포로 도입된다. 동시 형질전환은 경쇄 및 중쇄를 코딩하는 각각의 벡터 DNA를 동시에 숙주세포로 도입한 뒤 경쇄와 중쇄를 모두 발현하는 세포를 선별하는 방법이다. 표적 형질전환은 경쇄(또는 중쇄)를 포함하는 벡터로 형질전환 된 세포를 선별하고 경쇄를 발현하는 선별된 세포를 중쇄(또는 경쇄)를 포함하는 벡터로 다시 형질전환 하여 경쇄 및 중쇄 모두를 발현하는 세포를 최종적으로 선별하는 방법이다.The vector expressing the antibody of the present invention may be a vector system in which a light chain and a heavy chain are expressed simultaneously in one vector, or a system in which a light chain and a heavy chain are expressed in separate vectors, respectively. In the latter case, both vectors are introduced into host cells through co-transformation and targeted transformation. Simultaneous transformation is a method of selecting cells expressing both the light and heavy chains after simultaneously introducing each vector DNA encoding the light and heavy chains into a host cell. Target transformation involves selecting cells transformed with a vector containing a light chain (or heavy chain) and re-transforming the selected cells expressing the light chain with a vector containing a heavy chain (or light chain) to express both the light chain and the heavy chain. It is a method for final selection of cells.
또한, 본 발명은 재조합 발현벡터로 형질전환된 세포를 제공한다. In addition, the present invention provides a cell transformed with a recombinant expression vector.
본 발명의 벡터를 안정되면서 연속적으로 클로닝 및 발현시킬 수 있는 세포는 관련 기술 분야에 공지된 임의의 숙주 세포일 수 있으며, 예컨대, 에스케리치아 콜라이(Escherichia coli), 바실러스 서브틸리스 및 바실러스 츄린겐시스와 같은 바실러스 속 균주, 스트렙토마이세스(Streptomyces), 슈도모나스(Pseudomonas)(예를 들면, 슈도모나스 푸티다(Pseudomonas putida)), 프로테우스 미라빌리스(Proteus mirabilis) 또는 스타필로코쿠스(Staphylococcus)(예를 들면, 스타필로코쿠스 카르노수스(Staphylocus carnosus))와 같은 원핵 숙주 세포를 포함하나, 이에 제한되는 것은 아니다.Cells capable of stably and continuously cloning and expressing the vector of the present invention may be any host cell known in the art, for example, Escherichia coli, Bacillus subtilis and Bacillus thuringien. Bacillus sp. strains such as cis, Streptomyces, Pseudomonas (eg Pseudomonas putida), Proteus mirabilis or Staphylococcus (eg Pseudomonas putida) Examples include, but are not limited to, prokaryotic host cells such as Staphylocus carnosus).
상기 항체 또는 그의 항원 결합 단편의 제조 방법에서 형질전환 세포의 배양은 관련 기술 분야에 공지된 적당한 배지와 배양조건에 따라 이루어질 수 있다. 이러한 배양과정은 통상의 기술자라면 선택되는 균주에 따라 용이하게 조정하여 사용할 수 있다. 세포 배양은, 세포의 성장 방식에 따라 현탁배양과 부착배양, 배양방법에 따라 회분식, 유가식 및 연속배양식의 방법으로 구분된다. 배양에 사용되는 배지는 특정한 균주의 요구조건을 적절하게 만족시켜야 한다.In the method for producing the antibody or antigen-binding fragment thereof, the transformed cells can be cultured according to an appropriate medium and culture conditions known in the art. Such a culture process can be easily adjusted and used by those skilled in the art according to the selected strain. Cell culture is divided into suspension culture and adherent culture according to the cell growth method, and batch, fed-batch and continuous culture methods according to the culture method. The medium used for culture should suitably satisfy the requirements of a particular strain.
또한, 본 발명은 상기 항체 또는 그의 항원 결합 단편을 유효성분으로 포함하는 IsaA 항원 검출용 조성물을 제공한다.In addition, the present invention provides a composition for detecting IsaA antigen comprising the antibody or antigen-binding fragment thereof as an active ingredient.
본 발명에 있어서, "IsaA 항원"은 면역원성 포도상구균성 항원 A(immunodominant staphylococcal antigen A; IsaA)를 의미하는 것으로, IsaA는 높은 면역원성, 비공유성으로 세포벽에 결합하는 용해성 트랜스글리코실레이즈(transglycosylase)이다.In the present invention, "IsaA antigen" refers to immunogenic staphylococcal antigen A (IsaA), and IsaA is a highly immunogenic, non-covalent, soluble transglycosylase that binds to the cell wall. )to be.
또한, 본 발명은 상기 항체 또는 그의 항원 결합 단편을 유효성분으로 포함하는 황색포도상구균(Staphylococcus aureus) 검출용 조성물을 제공한다.In addition, the present invention provides a composition for detecting Staphylococcus aureus comprising the antibody or antigen-binding fragment thereof as an active ingredient.
또한, 본 발명은 항체 또는 그의 항원 결합 단편을 유효성분으로 포함하는 황색포도상구균(Staphylococcus aureus) 억제용 항균 조성물을 제공한다.In addition, the present invention provides an antibacterial composition for inhibiting Staphylococcus aureus comprising an antibody or antigen-binding fragment thereof as an active ingredient.
또한, 본 발명은 상기 항체 또는 그의 항원 결합 단편을 유효성분으로 포함하는 아토피 피부염 예방 또는 개선용 화장료 조성물을 제공한다. In addition, the present invention provides a cosmetic composition for preventing or improving atopic dermatitis comprising the antibody or antigen-binding fragment thereof as an active ingredient.
상기 화장료 조성물은 유효성분 외에 안정화제, 용해화제, 비타민, 안료 및 향료와 같은 통상적인 보조제, 그리고 담체를 포함할 수 있다.The cosmetic composition may include, in addition to the active ingredient, conventional adjuvants such as stabilizers, solubilizers, vitamins, pigments and fragrances, and carriers.
상기 화장료 조성물의 제형은 당업계에서 통상적으로 제조되는 어떠한 제형으로도 제조될 수 있으며, 피부외용연고, 크림, 유연화장수, 영양화장수, 팩, 에센스, 헤어토닉, 샴푸, 린스, 헤어 컨디셔너, 헤어 트리트먼트, 젤, 스킨 로션, 스킨소프너, 스킨토너, 아스트린젠트, 로션, 밀크로션, 모이스처 로션, 영양로션, 마사지 크림, 영양크림, 아이크림, 모이스처 크림, 핸드 크림, 파운데이션, 영양에센스, 선스크린, 비누, 클렌징폼, 클렌징로션, 클렌징크림, 바디 로션 및 바디 클렌저로 이루어지는 군으로부터 선택된 제형을 가질 수 있으나, 이에 제한되지 않는다. 이들 각 제형의 조성물은 그 제형의 제제화에 필요하고 적절한 각종의 기제와 첨가물을 함유할 수 있으며, 이들 성분의 종류와 양은 당업자에 의해 용이하게 선정될 수 있다.The formulation of the cosmetic composition may be prepared in any formulation conventionally prepared in the art, and external skin ointment, cream, softening lotion, nutrient lotion, pack, essence, hair tonic, shampoo, conditioner, hair conditioner, hair treatment ment, gel, skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nourishing lotion, massage cream, nourishing cream, eye cream, moisture cream, hand cream, foundation, nourishing essence, sunscreen, soap , cleansing foam, cleansing lotion, cleansing cream, body lotion and body cleanser may have a formulation selected from the group consisting of, but is not limited thereto. The composition of each of these formulations may contain various bases and additives necessary and appropriate for the formulation of the formulation, and the types and amounts of these components can be easily selected by those skilled in the art.
상기 제형이 페이스트, 크림 또는 겔인 경우에는 담체 성분으로서 동물성유, 식물성유, 왁스, 파라핀, 전분, 트라칸트, 셀룰로오스 유도체, 폴리에틸렌 글리콜, 실리콘, 벤토나이트, 실리카, 탈크 또는 산화아연 등이 이용될 수 있다.When the formulation is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as a carrier component. .
상기 제형이 파우더 또는 스프레이인 경우에는 담체 성분으로서 락토스, 탈크, 실리카, 알루미늄 히드록시드, 칼슘 실리케이트 또는 폴리아미드 파우더가 이용될 수 있고, 특히 스프레이인 경우에는 추가적으로 클로로플루오로히드로카본, 프로판/부탄 또는 디메틸 에테르와 같은 추진체를 포함할 수 있다.When the formulation is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component, and in particular, in the case of a spray, additional chlorofluorohydrocarbon, propane/butane or a propellant such as dimethyl ether.
상기 제형이 용액 또는 유탁액인 경우에는 담체 성분으로서 용매, 용해화제 또는 유탁화제가 이용되고, 예컨대 물, 에탄올, 이소프로판올, 에틸 카보네이트, 에틸 아세테이트, 벤질 알코올, 벤질 벤조에이트, 프로필렌 글리콜, 1,3-부틸글리콜 오일, 글리세롤 지방족 에스테르, 폴리에틸렌 글리콜 또는 소르비탄의 지방산 에스테르가 있다.When the formulation is a solution or emulsion, a solvent, solubilizer or emulsifier is used as a carrier component, for example, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3 -Butylglycol oil, glycerol fatty ester, polyethylene glycol or fatty acid ester of sorbitan.
상기 제형이 현탁액인 경우에는 담체 성분으로서 물, 에탄올 또는 프로필렌 글리콜과 같은 액상의 희석제, 에톡실화 이소스테아릴 알코올, 폴리옥시에틸렌 소르비톨 에스테르 및 폴리옥시에틸렌 소르비탄 에스테르와 같은 현탁제, 미소결정성 셀룰로오스, 알루미늄 메타히드록시드, 벤토나이트, 아가 또는 트라칸트 등이 이용될 수 있다.When the formulation is a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystalline cellulose , aluminum metahydroxide, bentonite, agar or tracanth may be used.
또한, 본 발명은 상기 항체 또는 그의 항원 결합 단편을 유효성분으로 포함하는 아토피 피부염 예방 또는 개선용 건강기능식품 조성물을 제공한다. In addition, the present invention provides a health functional food composition for preventing or improving atopic dermatitis comprising the antibody or antigen-binding fragment thereof as an active ingredient.
상기 건강기능식품 조성물은 분말, 과립, 정제, 캡슐, 시럽, 음료 또는 환의 형태로 제공될 수 있으며, 상기 건강식품조성물은 유효성분인 본 발명에 따른 조성물 이외에 다른 식품 또는 식품 첨가물과 함께 사용되고, 통상적인 방법에 따라 적절하게 사용될 수 있다. 유효성분의 혼합양은 그의 사용 목적 예를 들어 예방, 건강 또는 치료적 처치에 따라 적합하게 결정될 수 있다.The health functional food composition may be provided in the form of powder, granule, tablet, capsule, syrup, beverage or pill, and the health food composition is used together with other food or food additives in addition to the composition according to the present invention as an active ingredient, usually It may be appropriately used depending on the phosphorus method. The mixed amount of the active ingredient may be suitably determined according to the purpose of its use, for example, prophylactic, health or therapeutic treatment.
상기 건강기능식품 조성물에 함유된 항체 또는 그의 항원 결합 단편의 유효용량은 상기 약학조성물의 유효용량에 준해서 사용할 수 있으나, 건강 및 위생을 목적으로 하거나 또는 건강 조절을 목적으로 하는 장기간의 섭취의 경우에는 상기 범위 이하일 수 있으며, 유효성분은 안전성 면에서 아무런 문제가 없기 때문에 상기 범위 이상의 양으로도 사용될 수 있음은 확실하다.The effective dose of the antibody or antigen-binding fragment thereof contained in the health functional food composition can be used according to the effective dose of the pharmaceutical composition, but in the case of long-term intake for health and hygiene or health control may be less than the above range, and it is certain that the active ingredient can be used in an amount above the above range because there is no problem in terms of safety.
상기 건강식품의 종류에는 특별한 제한이 없고, 예로는 육류, 소세지, 빵, 쵸코렛, 캔디류, 스넥류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 차, 드링크제, 알콜 음료 및 비타민 복합제 등을 들 수 있다.The type of health food is not particularly limited, and examples include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, vitamin complexes, and the like.
또한, 본 발명은 상기 항체 또는 그의 항원 결합 단편을 유효성분으로 포함하는 아토피 피부염 예방 또는 치료용 약학조성물을 제공한다.In addition, the present invention provides a pharmaceutical composition for preventing or treating atopic dermatitis comprising the antibody or antigen-binding fragment thereof as an active ingredient.
본 발명의 약학 조성물은 약제학적으로 허용되는 담체를 추가로 포함할 수 있으며, 상기 약제학적으로 허용되는 담체는 제제시에 통상적으로 이용되는 것으로서, 락토스, 덱스트로스, 수크로스, 솔비톨, 만니톨, 전분, 아카시아 고무, 인산칼슘, 알기네이트, 젤라틴, 규산칼슘, 미세결정성 셀룰로오스, 폴리비닐피롤리돈, 셀룰로오스, 물, 시럽, 메틸셀룰로오스, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 활석, 스테아르산 마그네슘 및 미네랄 오일 등을 포함하나, 이에 한정되는 것은 아니다. 본 발명의 암 전이 예방 또는 치료용 조성물은 상기 성분들 이외에 윤활제, 습윤제, 감미제, 향미제, 유화제, 현탁제, 보존제 등을 추가로 포함할 수 있다. The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier, and the pharmaceutically acceptable carrier is commonly used in the formulation, and includes lactose, dextrose, sucrose, sorbitol, mannitol, starch. , Gum Acacia, Calcium Phosphate, Alginate, Gelatin, Calcium Silicate, Microcrystalline Cellulose, Polyvinylpyrrolidone, Cellulose, Water, Syrup, Methylcellulose, Methylhydroxybenzoate, Propylhydroxybenzoate, Talc, Stear acid magnesium and mineral oil, and the like. The composition for preventing or treating cancer metastasis of the present invention may further include a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like, in addition to the above components.
본 발명의 약학 조성물은 경구 또는 비경구로 투여할 수 있고, 비경구 투여인 경우에는 정맥내 주입, 피하주입, 근육 주입, 복강 주입, 내피 투여, 국소 투여, 비내 투여, 폐내 투여 및 직장 내 투여 등으로 투여할 수 있다. 경구 투여시, 단백질 또는 펩타이드는 소화가 되기 때문에 경구용 조성물은 활성 약제를 코팅하거나 위에서의 분해로부터 보호되도록 제형화 될 수 있으며, 본 발명의 조성물은 활성 물질이 표적 세포로 이동할 수 있는 임의의 장치에 의해 투여될 수 있다.The pharmaceutical composition of the present invention may be administered orally or parenterally, and in the case of parenteral administration, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration, intrapulmonary administration, rectal administration, etc. can be administered as When administered orally, since the protein or peptide is digested, oral compositions can be formulated to coat the active agent or to protect it from degradation in the stomach, and the composition of the present invention can be any device capable of transporting the active agent to a target cell. can be administered by
본 발명의 약학 조성물의 적합한 투여량은 제제화 방법, 투여 방식, 환자의 연령, 체중, 성별, 병적 상태, 음식, 투여 시간, 투여 경로, 배설 속도 및 반응 감응성과 같은 요인들에 의해 다양하며, 보통으로 숙련된 의사는 소망하는 치료 또는 예방에 효과적인 투여량을 용이하게 결정 및 처방할 수 있다. A suitable dosage of the pharmaceutical composition of the present invention varies depending on factors such as formulation method, administration mode, age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and response sensitivity of the patient, usually Thus, a skilled physician can easily determine and prescribe an effective dosage for the desired treatment or prevention.
본 발명의 약학 조성물은 당해 발명이 속하는 기술 분야에서 통상의 지식을 가진 자가 용이하게 실시할 수 있는 방법에 따라, 약제학적으로 허용되는 담체 및/또는 부형제를 이용하여 제제화하여 단위 용량 형태로 제조되거나 또는 다용량 용기 내에 내입시켜 제조될 수 있다. 이때 제형은 오일 또는 수성 매질중의 용액, 현탁액 또는 유화액 형태이거나 엑스제, 산제, 좌제, 분말제, 과립제, 정제 또는 캅셀제 형태일 수도 있으며, 분산제 또는 안정화제를 추가적으로 포함할 수 있다.The pharmaceutical composition of the present invention is formulated in a unit dosage form by using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by a person of ordinary skill in the art to which the present invention pertains. Alternatively, it may be prepared by being introduced into a multi-dose container. In this case, the formulation may be in the form of a solution, suspension or emulsion in oil or aqueous medium, or may be in the form of an extract, powder, suppository, powder, granule, tablet or capsule, and may additionally include a dispersing agent or stabilizer.
본 발명의 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고 종래의 치료제와는 순차적 또는 동시에 투여될 수 있다.The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 요지에 따라 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .
<< 실시예Example 1> 항- 1> clause- IsaAIsaA scFvscFv 항체 선별 Antibody screening
1. bio-panning 시행1. Implementation of bio-panning
7.6 × 109의 다양성을 가지는 OPAL library를 이용하여 bio-panning을 수행하였다. Epoxy magnetic bead에 10 μg의 IsaA 항원을 고정시키고 input phage를 반응시켰다. 항원과 반응한 phage를 용출(elution)하여 output titer를 측정하였다. 매 횟수마다 input과 output titer를 측정하여 bio-panning의 정보를 획득하고 정상적으로 시행되고 있는지를 확인하였다. 매 횟수마다 input phage는 1 × 1012 cfu/mL ≥의 phage를 사용하였다. output은 1차 - 6.5 × 106, 2차 - 1.13 × 104, 3차 - 4.21 × 107 cfu/mL으로 측정되었다. 따라서 bio-panning이 정상적으로 진행되었다는 것을 확인할 수 있었다.Bio-panning was performed using the OPAL library having a diversity of 7.6 × 10 9 . 10 μg of IsaA antigen was immobilized on an epoxy magnetic bead and the input phage was reacted. The output titer was measured by elution of the phage reacted with the antigen. By measuring the input and output titers every number of times, information of bio-panning was obtained and it was checked whether the operation was carried out normally. For each input phage, a phage of 1 × 10 12 cfu/mL ≥ was used. The output was measured as 1st - 6.5 × 10 6 , 2nd - 1.13 × 10 4 , 3rd - 4.21 × 10 7 cfu/mL. Therefore, it was confirmed that the bio-panning proceeded normally.
2. ELISA 분석2. ELISA analysis
고민감도, 고특이성 항체의 선별을 위해 ELISA 분석법을 수행하였다. 확보한 phage를 대장균에 감염시켜 항생제가 첨가된 LB plate에 도말하였다. 16 시간 동안 30℃ 배양기에서 배양한 후 생성된 콜로니를 랜덤하게 채집하였다. 각 콜로니를 LB 배지에 접종하여 배양한 후 IPTG를 처리하여 scFv 항체를 발현시키고 대장균을 용해(lysis)하여 수용성 분획(soluble fraction)을 취하여 ELISA를 수행하였다. 먼저, 50 ng의 IsaA 항원을 96-well ELISA plate에 고정시키고, 3% BSA가 함유된 PBS로 블락킹(blocking) 하였다. 1 시간 후, 위에서 얻은 세포 용해물(cell lysate)을 처리하여 37℃에서 2 시간 동안 반응시켰다. 0.1% Tween20가 함유된 PBS로 plate를 3회 씻어준 후 블락킹 용액(blocking solution)에 HRP-conjugated anti-HA 항체를 1:1000으로 희석하여 상온에서 1 시간 동안 반응시켰다. 0.1% Tween20가 함유된 PBS로 plate를 5회 씻어준 후 TMB substrate로 발색시켜 ELISA reader로 항원에 결합된 scFv 항체를 측정하였다. 96개의 항체를 분석하였고 OD 0.4를 양성 가이드라인(positive guideline), 0.1을 음성 가이드라인(negative guideline)으로 임의로 정하여 10종을 선별하였다(도 1). An ELISA assay was performed for the selection of highly sensitive and highly specific antibodies. The obtained phage was infected with E. coli and spread on an LB plate to which antibiotics were added. After culturing in an incubator at 30° C. for 16 hours, the resulting colonies were randomly collected. After each colony was inoculated in LB medium and cultured, IPTG was treated to express scFv antibody, and Escherichia coli was lysed to obtain a soluble fraction, followed by ELISA. First, 50 ng of IsaA antigen was immobilized on a 96-well ELISA plate, and blocked with PBS containing 3% BSA. After 1 hour, the cell lysate obtained above was treated and reacted at 37° C. for 2 hours. After washing the
<< 실시예Example 2> 항- 2> Paragraph- IsaAIsaA scFvscFv 항체 서열 분석 antibody sequencing
ELISA 분석을 통하여 선별된 IsaA scFv 10종의 양성 클론(positive clone)에 대하여 서열분석(sequencing)을 통해 개별적 클론(clone)을 선별하였다. 앞서 선별한 양성 클론 중에 중복되는 클론이 존재할 가능성이 있기 때문에 서열분석이 필요하였다. 서열 분석 결과, 10 종의 양성 클론 중 7종이 개별적인 클론임을 확인하였다.Individual clones were selected through sequencing with respect to 10 positive clones of IsaA scFv selected through ELISA analysis. Since there is a possibility that overlapping clones exist among the previously selected positive clones, sequencing was required. As a result of sequence analysis, it was confirmed that 7 out of 10 positive clones were individual clones.
<< 실시예Example 3> 선별된 항- 3> selected anti- IsaAIsaA scFvscFv 항체 정제 및 민감도 분석 Antibody purification and sensitivity analysis
1. 항-1. Anti- IsaAIsaA (G4) (G4) scFvscFv 항체 정제 Antibody purification
선별된 7종 중 발현율이 높은 항-IsaA (G4) scFv 항체(표 1)를 정제하였다. 항-IsaA scFv 클론(G4)이 형질전환(transformation) 되어 있는 ER2738 E. coli cell을 500 mL의 SB media에 배양시킨 후 1 × TES buffer를 이용하여 세포를 용해(lysis) 시켰다. 세포 용해물(cell lysate)을 0.5 mL Ni bead에 반응시키고 이미다졸(imidazole)을 이용하여 용출(elution) 하였다.Anti-IsaA (G4) scFv antibody (Table 1) having a high expression rate among the selected 7 types was purified. ER2738 E. coli cells transformed with anti-IsaA scFv clone (G4) were cultured in 500 mL of SB media and then lysed using 1 × TES buffer. The cell lysate was reacted with 0.5 mL Ni beads and eluted using imidazole.
2. 정제된 항-2. Purified anti- IsaAIsaA (G4) (G4) scFvscFv 항체의 민감도 분석 Antibody sensitivity analysis
정제된 항-IsaA (G4) scFv 항체의 민감도 분석을 시행하였다. 민감도 분석은 ELISA 분석법을 이용하였다. 우선 여러 농도의 IsaA 항원을 ELISA plate에 고정시킨 후 2 μg/mL의 항-IsaA (G4) scFv 항체를 처리하였다. 분석은 1:1000으로 희석한 HRP-conjugated anti-HA 항체를 이용하여 분석하였다. 그 결과, 항-IsaA (G4) scFv 항체의 EC50은 24.266 μg/mL, LOD는 0.04 μg/mL로 분석되었다(도 2).Sensitivity analysis of purified anti-IsaA (G4) scFv antibody was performed. Sensitivity analysis was performed using an ELISA assay. First, various concentrations of IsaA antigen were immobilized on an ELISA plate and then treated with anti-IsaA (G4) scFv antibody at 2 μg/mL. Analysis was performed using HRP-conjugated anti-HA antibody diluted 1:1000. As a result, the anti-IsaA (G4) scFv antibody had an EC50 of 24.266 μg/mL and an LOD of 0.04 μg/mL ( FIG. 2 ).
<< 실시예Example 4> 황색포도상구균을 이용한 항- 4> Anti- Staphylococcus aureus IsaAIsaA (G4) (G4) scFvscFv 항체의 활성도 분석 Antibody activity analysis
항-IsaA (G4) scFv 항체의 실제 검체인 황색포도상구균(Staphylococcus aureus)과의 반응성을 확인하기 위해 ELISA 분석법을 이용하였다. 박테리아 세포를 96-well plate에 각 well 당 101~106 개씩 분주하여 4℃에서 16 시간 부착시켰다. 부착된 박테리아 세포를 3% BSA가 포함된 PBS를 이용하여 블락킹(blocking)하고, 각 well에 정제한 항-IsaA (G4) scFv 항체를 2 μg/mL로 처리한 후 37℃에서 2 시간 동안 반응시켰다. 0.05% Tween20가 포함된 PBS로 세척하여 반응하지 않은 항체를 제거하고, HRP-conjugated anti-HA 항체를 1:1000 비율로 blocking solution에 희석하여 처리한 다음 37℃에서 1 시간 동안 반응시켰다. 세척 후 TMB 기질 수용액을 이용하여 상온에서 15 분간 발색반응을 진행한 다음 2.5 N 황산용액을 첨가하여 반응을 종료 시키고 ELISA reader를 이용하여 450 nm에서 흡광도를 측정하였다. 분석에서 음성대조구로 Lactobacillus rhamnosus를 이용하였다(도 3). 그 결과, 항-IsaA (G4) scFv 항체는 황색포도상구균에 특이적으로 결합하는 것을 확인하였다.An ELISA assay was used to confirm the reactivity of the anti-IsaA (G4) scFv antibody with Staphylococcus aureus, an actual sample. Bacterial cells were aliquoted in each well of 10 1 to 10 6 in a 96-well plate and adhered at 4°C for 16 hours. The adhered bacterial cells were blocked using PBS containing 3% BSA, and each well was treated with 2 μg/mL of purified anti-IsaA (G4) scFv antibody, and then at 37° C. for 2 hours. reacted. The unreacted antibody was removed by washing with PBS containing 0.05% Tween20, and the HRP-conjugated anti-HA antibody was diluted in a blocking solution at a ratio of 1:1000 and treated, followed by reaction at 37°C for 1 hour. After washing, the color reaction was performed at room temperature for 15 minutes using an aqueous TMB substrate solution, and then 2.5 N sulfuric acid solution was added to terminate the reaction, and absorbance was measured at 450 nm using an ELISA reader. In the analysis, Lactobacillus rhamnosus was used as a negative control ( FIG. 3 ). As a result, it was confirmed that the anti-IsaA (G4) scFv antibody specifically binds to Staphylococcus aureus.
<< 실시예Example 5> 세포투과성 항- 5> Cell-permeable anti- IsaAIsaA (G4) (G4) scFvscFv 항체 제작 Antibody production
1. TAT-항-1. TAT-anti- IsaAIsaA (G4) (G4) scFvscFv DNA construct 제작 DNA construct production
앞서 선별된 항-IsaA scFv를 primer를 이용하여 제한효소 사이트(restriction enzyme site)와 TAT을 삽입한 PCR 산물을 생산하였다. PCR 산물(insert)와 pET28a(+) vector를 EcoR I 1 μL, Xho I 1 μL 처리하여 37℃에서 한 시간 동안 반응시킨 후 정제하였다. 정제된 vector와 insert의 농도를 nanodrop으로 측정하고 vector와 insert의 비율별로 T4 ligase를 이용하여 상온에서 16 시간 동안 ligation을 진행하였다. Ligation이 끝난 후 DH5α에 형질전환 한 후 카나마이신이 첨가된 LB plate에 도말하여 37℃에서 16 시간 동안 배양하고 다음 날 콜로니를 취하여 카나마이신이 첨가된 LB media에 접종하여 37℃에서 16 시간 동안 배양하였다. 다음 날 insert가 삽입되었는지 확인하기 위해 플라스미드(plasmid)를 분리하여 EcoR I과 Xho I으로 절단(restriction)하고, 1% 아가로스 젤(agarose gel)에 전기영동하여 밴드를 확인하였다. A PCR product was produced in which a restriction enzyme site and TAT were inserted using the previously selected anti-IsaA scFv primer. The PCR product (insert) and pET28a(+) vector were treated with EcoR I 1 μL and
2. TAT-항-2. TAT-anti- IsaAIsaA (G4) (G4) scFvscFv 항체 정제 Antibody purification
클로닝 된 TAT-항-IsaA (G4) scFv 항체 클론을 BL21(DE3) 대장균 숙주에 형질전환 시켰다. 형질전환체는 카나마이신이 포함된 LB 배지에서 OD600 값이 0.6이 될 때까지 배양하고, 16℃에서 0.5 mM IPTG를 처리하여 발현 유도(induction) 하였다. 배양액을 원심분리하여 얻은 세포들을 lysis buffer(50 mM Tris-Hcl, pH 7.5, 150 mM NaCl)에 현탁한 후 초음파 분쇄기를 이용하여 파쇄한 다음 원심분리하여 상층액을 얻었다. 유도 발현된 단백질만을 순수 분리 정제하기 위해 Ni-NTA에 친화력을 갖는 레진을 사용하였으며, 정제된 단백질은 SDS-PAGE 분석을 통하여 확인하였다(도 4).The cloned TAT-anti-IsaA (G4) scFv antibody clone was transformed into a BL21 (DE3) E. coli host. Transformants were cultured in LB medium containing kanamycin until the OD 600 value reached 0.6, and expression was induced by treatment with 0.5 mM IPTG at 16°C. Cells obtained by centrifugation of the culture medium were suspended in lysis buffer (50 mM Tris-Hcl, pH 7.5, 150 mM NaCl), disrupted using an ultrasonicator, and then centrifuged to obtain a supernatant. A resin having an affinity for Ni-NTA was used to purely separate and purify only the induced expressed protein, and the purified protein was confirmed through SDS-PAGE analysis (FIG. 4).
<< 실시예Example 6> 세포투과성 항- 6> Cell-permeable anti- IsaAIsaA (G4) (G4) scFvscFv 항체의 세포 투과 분석 Cell Permeation Assays of Antibodies
TAT-항-IsaA (G4) scFv 항체의 세포 투과를 확인하기 위하여 웨스턴 블랏 분석법을 이용하였다. 인간 피부 세포를 6-well 플레이트에 1 × 106 개로 분주한 후 10% FBS(fetal bovine serum), 페니실린(100 U/mL), 스트렙토마이신(100 μg/mL)을 함유하는 RPMI 배지를 넣고 37℃를 유지하여 5% 이산화탄소를 포함하는 배양기에서 배양하였다. 배양 후 FBS가 포함되지 않은 1.5 mL의 신선한 배양액으로 교체하고 여러 농도의 TAT-항-IsaA (G4) scFv를 처리하였다. 처리 1 시간 뒤, 세포를 PBS로 충분히 세척한 다음 회수하여 TAT-항-IsaA (G4) scFv의 세포 내 투과된 정도를 웨스턴 블랏 분석으로 확인하였다. 세포 내 투과된 TAT-항-IsaA (G4) scFv가 정확한 위치(크기)에서 확인되는지를 알아보기 위하여, 순수 정제된 TAT-항-IsaA (G4) scFv 항체(Control)도 함께 전기영동하였다. 도 5에 나타낸 바와 같이, TAT-항-IsaA (G4) scFv는 농도 의존적으로 세포 내 투과가 효과적으로 일어났음을 확인하였다.Western blot analysis was used to confirm cell permeation of TAT-anti-IsaA (G4) scFv antibody. After dividing human skin cells into 1 × 10 6 cells in a 6-well plate, RPMI medium containing 10% fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (100 μg/mL) was added and 37 ℃ was maintained and cultured in an incubator containing 5% carbon dioxide. After incubation, it was replaced with 1.5 mL of fresh culture solution without FBS and treated with TAT-anti-IsaA (G4) scFv at various concentrations. After 1 hour of treatment, the cells were sufficiently washed with PBS and recovered, and the degree of intracellular permeation of TAT-anti-IsaA (G4) scFv was confirmed by Western blot analysis. In order to determine whether the intracellularly permeated TAT-anti-IsaA (G4) scFv was identified at the correct location (size), pure purified TAT-anti-IsaA (G4) scFv antibody (Control) was also electrophoresed. As shown in FIG. 5 , it was confirmed that TAT-anti-IsaA (G4) scFv had effective intracellular permeation in a concentration-dependent manner.
<< 실시예Example 7> 세포투과성 항- 7> Cell-permeable anti- IsaAIsaA (G4) (G4) scFvscFv 세포독성 시험 Cytotoxicity test
TAT-항-IsaA (G4) scFv 항체에 대한 세포독성을 알아보기 위하여, 인간 피부 세포를 96-well 플레이트에 well 당 1 × 104 개로 분주한 후 37℃를 유지하여 5% 이산화탄소를 포함하는 배양기에서 배양하였다. 24 시간 후, TAT-항-IsaA (G4) scFv를 농도별로 처리한 다음 같은 배양 조건에서 추가 배양하였다. 배양이 끝난 후 MTT{3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide} 용액을 넣고 3 시간 동안 반응시킨 다음 배양액을 제거하고 DMSO(Dimethyl sulfoxide)를 100 μL씩 넣어주었다. 15 분간 흔들어 준 다음 ELISA reader로 570 nm에서 흡광도를 측정하였다. 표 2에 나타낸 바와 같이, TAT-항-IsaA (G4) scFv를 0.1, 1, 10, 50, 100, 200 ppm 농도로 처리했을 때, 200 ppm 농도까지 세포모양 변형 및 세포 생존율의 변화가 거의 없음을 확인하였다. 따라서, TAT-항-IsaA (G4) scFv 항체의 200 ppm 농도까지는 세포 독성이 없는 물질임을 알 수 있었다.To investigate the cytotoxicity of TAT-anti-IsaA (G4) scFv antibody, human skin cells were aliquoted into 1 × 10 4 per well in a 96-well plate and maintained at 37°C in an incubator containing 5% carbon dioxide. cultured in After 24 hours, TAT-anti-IsaA (G4) scFv was treated by concentration and then further cultured under the same culture conditions. After incubation, MTT{3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide} solution was added and reacted for 3 hours, then the culture solution was removed and DMSO (dimethyl sulfoxide) was added 100 μL each. gave. After shaking for 15 minutes, absorbance was measured at 570 nm with an ELISA reader. As shown in Table 2, when TAT-anti-IsaA (G4) scFv was treated at concentrations of 0.1, 1, 10, 50, 100, and 200 ppm, there was little change in cell shape transformation and cell viability up to 200 ppm concentration. was confirmed. Therefore, it was confirmed that the TAT-anti-IsaA (G4) scFv antibody was not cytotoxic up to a concentration of 200 ppm.
<< 실시예Example 8> 세포투과성 항- 8> cell-permeable anti- IsaAIsaA (G4) (G4) scFv의scFv 황색포도상구균 항균력 시험 Staphylococcus aureus antibacterial activity test
황색포도상구균(Staphylococcus aureus)의 성장에 대한 TAT-항-IsaA (G4) scFv 항체의 효과를 알아보았다. 균주를 TSB 배지(Tryptic soy broth)에 접종하여 OD 0.1이 될 때까지 배양한 후 TAT-항-IsaA (G4) scFv 항체를 처리하였다. 37℃에서 24 시간 배양하면서 시간대별로 600 nm에서 흡광도를 측정하여, 황색포도상구균의 성장능을 조사하였다. 음성대조구로 Lactobacillus rhamnosus를 이용하였다. 표 3에 나타낸 바와 같이, TAT-항-IsaA (G4) scFv는 황색포도상구균의 성장을 저해하는 것을 확인하였다.The effect of TAT-anti-IsaA (G4) scFv antibody on the growth of Staphylococcus aureus was investigated. The strain was inoculated in TSB medium (Tryptic soy broth) and cultured until OD 0.1 was treated with TAT-anti-IsaA (G4) scFv antibody. The growth ability of Staphylococcus aureus was investigated by measuring the absorbance at 600 nm for each time period while culturing at 37° C. for 24 hours. Lactobacillus rhamnosus was used as a negative control. As shown in Table 3, it was confirmed that TAT-anti-IsaA (G4) scFv inhibited the growth of Staphylococcus aureus.
(ppm)(ppm)
이상으로 본 발명의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적인 기술은 단지 바람직한 구현 예일 뿐이며, 이에 본 발명의 범위가 제한되는 것이 아닌 점은 명백하다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항과 그의 등가물에 의하여 정의된다고 할 것이다.As the specific parts of the present invention have been described in detail above, for those of ordinary skill in the art, these specific techniques are only preferred embodiments, and it is clear that the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.
<110> HAUUL BIO <120> Anti-IsaA G4 antibody inhibiting Staphylococcus aureus and use thereof <130> DP-2019-0270 <160> 8 <170> KopatentIn 2.0 <210> 1 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> light chain CDR1 <400> 1 Thr Gly Ser Ser Ser Asn Ile Gly Asn Asn Ala Val Tyr 1 5 10 <210> 2 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> light chain CDR2 <400> 2 Ser Asn Ser Gln 1 <210> 3 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> light chain CDR3 <400> 3 Gly Ala Trp Asp Ala Ser Leu Ser Ala Tyr Val 1 5 10 <210> 4 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> heavy chain CDR1 <400> 4 Gly Tyr Ala Met Ser 1 5 <210> 5 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> heavy chain CDR2 <400> 5 Val Ile Ser Ser Asp Gly Gly Ser Thr 1 5 <210> 6 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> heavy chain CDR3 <400> 6 Lys Ile Thr Arg Lys Phe Asp Tyr 1 5 <210> 7 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> TAT peptide <400> 7 Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg 1 5 10 <210> 8 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> TAT peptide <400> 8 tatggccgca aaaaacgccg ccagcgccgc cgc 33 <110> HAUUL BIO <120> Anti-IsaA G4 antibody inhibiting Staphylococcus aureus and use it <130> DP-2019-0270 <160> 8 <170> KopatentIn 2.0 <210> 1 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> light chain CDR1 <400> 1 Thr Gly Ser Ser Ser Asn Ile Gly Asn Asn Ala Val Tyr 1 5 10 <210> 2 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> light chain CDR2 <400> 2 Ser Asn Ser Gln One <210> 3 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> light chain CDR3 <400> 3 Gly Ala Trp Asp Ala Ser Leu Ser Ala Tyr Val 1 5 10 <210> 4 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> heavy chain CDR1 <400> 4 Gly Tyr Ala Met Ser 1 5 <210> 5 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> heavy chain CDR2 <400> 5 Val Ile Ser Ser Asp Gly Gly Ser Thr 1 5 <210> 6 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> heavy chain CDR3 <400> 6 Lys Ile Thr Arg Lys Phe Asp Tyr 1 5 <210> 7 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> TAT peptide <400> 7 Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg 1 5 10 <210> 8 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> TAT peptide <400> 8 tatggccgca aaaaacgccg ccagcgccgc cgc 33
Claims (11)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020190154518A KR102341805B1 (en) | 2019-11-27 | 2019-11-27 | Anti-IsaA G4 antibody specific to Staphylococcus aureus and use thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020190154518A KR102341805B1 (en) | 2019-11-27 | 2019-11-27 | Anti-IsaA G4 antibody specific to Staphylococcus aureus and use thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| KR20210066072A KR20210066072A (en) | 2021-06-07 |
| KR102341805B1 true KR102341805B1 (en) | 2021-12-22 |
Family
ID=76374105
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| KR1020190154518A KR102341805B1 (en) | 2019-11-27 | 2019-11-27 | Anti-IsaA G4 antibody specific to Staphylococcus aureus and use thereof |
Country Status (1)
| Country | Link |
|---|---|
| KR (1) | KR102341805B1 (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2009031009A (en) | 2007-07-24 | 2009-02-12 | St Marianna Univ School Of Medicine | Method for detecting or identifying staphylococcus aureus, and detection kit |
| US20150299655A1 (en) | 2012-11-27 | 2015-10-22 | Commissariat A L'energie Atomique Et Aux Energies Alternatives | Method for obtaining antigen-specific human antibodies by in vitro immunisation |
| JP2016047828A (en) | 2009-05-18 | 2016-04-07 | ユリウス・マクシミリアンス−ウニヴェルジテート・ヴュルツブルクＪｕｌｉｕｓ Ｍａｘｉｍｉｌｉａｎｓ−Ｕｎｉｖｅｒｓｉｔａｅｔ Ｗｕｅｒｚｂｕｒｇ | ANTIBODIES OR FRAGMENTS THEREOF DIRECTED AGAINST STAPHYLOCOCCUS AUREUS EPITOPE IsaA OR IsaB |
| US20170002063A1 (en) * | 2013-12-13 | 2017-01-05 | Rijksuniversiteit Groningen | Antibodies against staphylococcus aureus and uses therof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP5439127B2 (en) * | 2009-11-13 | 2014-03-12 | 株式会社さいわいメディカル | Treatment for psoriasis or atopic dermatitis |
-
2019
- 2019-11-27 KR KR1020190154518A patent/KR102341805B1/en active IP Right Grant
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2009031009A (en) | 2007-07-24 | 2009-02-12 | St Marianna Univ School Of Medicine | Method for detecting or identifying staphylococcus aureus, and detection kit |
| JP2016047828A (en) | 2009-05-18 | 2016-04-07 | ユリウス・マクシミリアンス−ウニヴェルジテート・ヴュルツブルクＪｕｌｉｕｓ Ｍａｘｉｍｉｌｉａｎｓ−Ｕｎｉｖｅｒｓｉｔａｅｔ Ｗｕｅｒｚｂｕｒｇ | ANTIBODIES OR FRAGMENTS THEREOF DIRECTED AGAINST STAPHYLOCOCCUS AUREUS EPITOPE IsaA OR IsaB |
| US20150299655A1 (en) | 2012-11-27 | 2015-10-22 | Commissariat A L'energie Atomique Et Aux Energies Alternatives | Method for obtaining antigen-specific human antibodies by in vitro immunisation |
| US20170002063A1 (en) * | 2013-12-13 | 2017-01-05 | Rijksuniversiteit Groningen | Antibodies against staphylococcus aureus and uses therof |
Also Published As
| Publication number | Publication date |
|---|---|
| KR20210066072A (en) | 2021-06-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| van den Berg et al. | A human monoclonal antibody targeting the conserved staphylococcal antigen IsaA protects mice against Staphylococcus aureus bacteremia | |
| DK2668208T3 (en) | The cross-reactive antibody Staphylococcus Aureus | |
| KR20190112003A (en) | Antibody Molecular-Drug Conjugates and Uses thereof | |
| KR102017240B1 (en) | Anti-VAMP2 antibodies inhibiting SNARE complex and use thereof | |
| KR20170136637A (en) | Anti-Staphylococcus aureus antibody formulation | |
| KR20100028558A (en) | Pseudomonas aeruginosa outer membrane protein pa4710 | |
| EP2999713A2 (en) | Generation of highly potent antibodies neutralizing the lukgh (lukab) toxin of staphylococcus aureus | |
| KR102341805B1 (en) | Anti-IsaA G4 antibody specific to Staphylococcus aureus and use thereof | |
| JP2013538573A (en) | Staphylococcus aureus-specific human recombinant polyclonal antibodies and uses thereof | |
| KR102341804B1 (en) | Anti-IsaA F10 antibody inhibiting Staphylococcus aureus and use thereof | |
| WO2014061783A1 (en) | Human antibody specific to toxin produced from clostridium difficile, or antigen-binding fragment thereof | |
| Wang et al. | Identification of a conserved linear B-cell epitope in the Staphylococcus aureus GapC protein | |
| KR20080068048A (en) | Outer coat protein pa5158 of pseudomonas aeruginosa | |
| KR102297991B1 (en) | Anti-Syntaxin 1A antibodies inhibiting SNARE complex and use thereof | |
| KR102274841B1 (en) | Anti-SNAP 25 antibodies inhibiting SNARE complex and use thereof | |
| WO2014160098A2 (en) | Bordetella specific human recombinant antibodies and uses thereof | |
| KR102638640B1 (en) | Use of anti-IsaA F10 antibody for preventing or improving atopic dermatitis | |
| KR102494042B1 (en) | Anti-DKK-1 antibody promoting the growth of human dermal papilla cells and use thereof | |
| KR20230150518A (en) | Anti-SNAP 25 B6 antibodies for preventing and improving skin wrinkle and use thereof | |
| KR102236127B1 (en) | Anti-tyrosinase antibodies inhibiting tyrosinase and use thereof | |
| KR20170137111A (en) | s. Antibodies directed against immunoglobulin binding proteins of Aureus | |
| KR102180731B1 (en) | Antibodies for neutralizing anthrax toxin and pharmaceutical composition comprising the same | |
| KR20220070116A (en) | Anti-tyrosinase B8 antibody that inhibits melanogenesis and use thereof | |
| CA2355831A1 (en) | Novel fibronectin-binding protein | |
| CN117396181A (en) | Use of anti-IsaA F10 antibodies for the prevention and amelioration of atopic dermatitis |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| E902 | Notification of reason for refusal | ||
| AMND | Amendment | ||
| E601 | Decision to refuse application | ||
| AMND | Amendment | ||
| X701 | Decision to grant (after re-examination) |