WO2023037329A1 - Synthetic protein for use in the medical field - Google Patents
Synthetic protein for use in the medical field Download PDFInfo
- Publication number
- WO2023037329A1 WO2023037329A1 PCT/IB2022/058567 IB2022058567W WO2023037329A1 WO 2023037329 A1 WO2023037329 A1 WO 2023037329A1 IB 2022058567 W IB2022058567 W IB 2022058567W WO 2023037329 A1 WO2023037329 A1 WO 2023037329A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- protein
- seq
- sequence
- sequence identity
- pharmaceutically acceptable
- Prior art date
Links
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 96
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 91
- 239000000203 mixture Substances 0.000 claims description 30
- 241000588724 Escherichia coli Species 0.000 claims description 17
- 238000000034 method Methods 0.000 claims description 17
- 241000235058 Komagataella pastoris Species 0.000 claims description 16
- 241000700605 Viruses Species 0.000 claims description 16
- 239000000645 desinfectant Substances 0.000 claims description 13
- 239000000499 gel Substances 0.000 claims description 10
- 239000006166 lysate Substances 0.000 claims description 9
- 239000000969 carrier Substances 0.000 claims description 8
- 244000005700 microbiome Species 0.000 claims description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- 239000007787 solid Substances 0.000 claims description 6
- 239000007921 spray Substances 0.000 claims description 6
- 239000000872 buffer Substances 0.000 claims description 5
- 239000001913 cellulose Substances 0.000 claims description 5
- 229920002678 cellulose Polymers 0.000 claims description 5
- 239000007788 liquid Substances 0.000 claims description 5
- 239000011780 sodium chloride Substances 0.000 claims description 5
- 238000011282 treatment Methods 0.000 claims description 5
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 claims description 4
- 239000002671 adjuvant Substances 0.000 claims description 4
- 239000006071 cream Substances 0.000 claims description 4
- 239000003349 gelling agent Substances 0.000 claims description 4
- 229920000609 methyl cellulose Polymers 0.000 claims description 4
- 239000001923 methylcellulose Substances 0.000 claims description 4
- 235000010981 methylcellulose Nutrition 0.000 claims description 4
- 239000000137 peptide hydrolase inhibitor Substances 0.000 claims description 4
- 239000003443 antiviral agent Substances 0.000 claims description 3
- 230000001580 bacterial effect Effects 0.000 claims description 3
- 230000008030 elimination Effects 0.000 claims description 3
- 238000003379 elimination reaction Methods 0.000 claims description 3
- 230000002538 fungal effect Effects 0.000 claims description 3
- 150000002632 lipids Chemical class 0.000 claims description 3
- 235000013336 milk Nutrition 0.000 claims description 3
- 210000004080 milk Anatomy 0.000 claims description 3
- 239000000546 pharmaceutical excipient Substances 0.000 claims description 3
- 239000000725 suspension Substances 0.000 claims description 3
- 239000002562 thickening agent Substances 0.000 claims description 3
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims description 2
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 claims description 2
- 235000010443 alginic acid Nutrition 0.000 claims description 2
- 229920000615 alginic acid Polymers 0.000 claims description 2
- 229910052791 calcium Inorganic materials 0.000 claims description 2
- 235000010410 calcium alginate Nutrition 0.000 claims description 2
- 239000000648 calcium alginate Substances 0.000 claims description 2
- 239000002502 liposome Substances 0.000 claims description 2
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims description 2
- 239000008267 milk Substances 0.000 claims description 2
- 235000010413 sodium alginate Nutrition 0.000 claims description 2
- 239000000661 sodium alginate Substances 0.000 claims description 2
- 229940005550 sodium alginate Drugs 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims 4
- 239000003937 drug carrier Substances 0.000 claims 2
- 208000036142 Viral infection Diseases 0.000 claims 1
- 230000009385 viral infection Effects 0.000 claims 1
- 235000018102 proteins Nutrition 0.000 description 76
- 210000004027 cell Anatomy 0.000 description 18
- 239000000243 solution Substances 0.000 description 15
- 101100083082 Pyrus communis PGIP gene Proteins 0.000 description 13
- 239000013598 vector Substances 0.000 description 13
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 9
- 108020004414 DNA Proteins 0.000 description 9
- 150000001413 amino acids Chemical class 0.000 description 9
- 238000000746 purification Methods 0.000 description 9
- 241000894006 Bacteria Species 0.000 description 8
- 101710198474 Spike protein Proteins 0.000 description 8
- 238000004659 sterilization and disinfection Methods 0.000 description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 8
- 238000002474 experimental method Methods 0.000 description 7
- 210000004379 membrane Anatomy 0.000 description 7
- 239000012528 membrane Substances 0.000 description 7
- 239000013612 plasmid Substances 0.000 description 7
- 238000002360 preparation method Methods 0.000 description 7
- 238000006243 chemical reaction Methods 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 238000004520 electroporation Methods 0.000 description 6
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 6
- 230000003612 virological effect Effects 0.000 description 6
- 241000196324 Embryophyta Species 0.000 description 5
- 108090000288 Glycoproteins Proteins 0.000 description 5
- 102000003886 Glycoproteins Human genes 0.000 description 5
- 108010059820 Polygalacturonase Proteins 0.000 description 5
- 101000629318 Severe acute respiratory syndrome coronavirus 2 Spike glycoprotein Proteins 0.000 description 5
- 230000000840 anti-viral effect Effects 0.000 description 5
- 230000006870 function Effects 0.000 description 5
- 108020003175 receptors Proteins 0.000 description 5
- 102000005962 receptors Human genes 0.000 description 5
- 238000012546 transfer Methods 0.000 description 5
- 241001678559 COVID-19 virus Species 0.000 description 4
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 4
- 229920002230 Pectic acid Polymers 0.000 description 4
- 229920001213 Polysorbate 20 Polymers 0.000 description 4
- 229940096437 Protein S Drugs 0.000 description 4
- 238000010367 cloning Methods 0.000 description 4
- 230000007613 environmental effect Effects 0.000 description 4
- 230000004927 fusion Effects 0.000 description 4
- 230000003993 interaction Effects 0.000 description 4
- 235000005772 leucine Nutrition 0.000 description 4
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 4
- 241000894007 species Species 0.000 description 4
- 230000000699 topical effect Effects 0.000 description 4
- 241000711573 Coronaviridae Species 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 101710116124 Polygalacturonase inhibitor Proteins 0.000 description 3
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 3
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 3
- 235000019892 Stellar Nutrition 0.000 description 3
- 240000006365 Vitis vinifera Species 0.000 description 3
- 235000014787 Vitis vinifera Nutrition 0.000 description 3
- 230000009471 action Effects 0.000 description 3
- 239000000443 aerosol Substances 0.000 description 3
- 230000003321 amplification Effects 0.000 description 3
- 230000005540 biological transmission Effects 0.000 description 3
- 210000000170 cell membrane Anatomy 0.000 description 3
- 230000009089 cytolysis Effects 0.000 description 3
- 230000006378 damage Effects 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 235000013305 food Nutrition 0.000 description 3
- 235000002532 grape seed extract Nutrition 0.000 description 3
- 208000015181 infectious disease Diseases 0.000 description 3
- 229930027917 kanamycin Natural products 0.000 description 3
- 229960000318 kanamycin Drugs 0.000 description 3
- 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 3
- 229930182823 kanamycin A Natural products 0.000 description 3
- 230000007246 mechanism Effects 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 108020004410 pectinesterase Proteins 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 238000011321 prophylaxis Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 210000002845 virion Anatomy 0.000 description 3
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- 101710163270 Nuclease Proteins 0.000 description 2
- 108091005804 Peptidases Proteins 0.000 description 2
- 239000004365 Protease Substances 0.000 description 2
- 108010084455 Zeocin Proteins 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 150000007513 acids Chemical class 0.000 description 2
- 235000001014 amino acid Nutrition 0.000 description 2
- 230000000845 anti-microbial effect Effects 0.000 description 2
- 238000004061 bleaching Methods 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 238000009792 diffusion process Methods 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 238000005538 encapsulation Methods 0.000 description 2
- 210000003527 eukaryotic cell Anatomy 0.000 description 2
- 238000006206 glycosylation reaction Methods 0.000 description 2
- WQYVRQLZKVEZGA-UHFFFAOYSA-N hypochlorite Chemical compound Cl[O-] WQYVRQLZKVEZGA-UHFFFAOYSA-N 0.000 description 2
- 239000004615 ingredient Substances 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 239000006210 lotion Substances 0.000 description 2
- 239000011777 magnesium Substances 0.000 description 2
- 229910052749 magnesium Inorganic materials 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 239000002324 mouth wash Substances 0.000 description 2
- CWCMIVBLVUHDHK-ZSNHEYEWSA-N phleomycin D1 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@@H](N=1)C=1SC=C(N=1)C(=O)NCCCCNC(N)=N)[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 CWCMIVBLVUHDHK-ZSNHEYEWSA-N 0.000 description 2
- 239000010318 polygalacturonic acid Substances 0.000 description 2
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 239000012146 running buffer Substances 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- 239000002884 skin cream Substances 0.000 description 2
- 239000000344 soap Substances 0.000 description 2
- 238000000527 sonication Methods 0.000 description 2
- 238000005507 spraying Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 239000000606 toothpaste Substances 0.000 description 2
- 238000013518 transcription Methods 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- 239000013638 trimer Substances 0.000 description 2
- 210000000605 viral structure Anatomy 0.000 description 2
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- NVKAWKQGWWIWPM-ABEVXSGRSA-N 17-β-hydroxy-5-α-Androstan-3-one Chemical compound C1C(=O)CC[C@]2(C)[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CC[C@H]21 NVKAWKQGWWIWPM-ABEVXSGRSA-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
- TWJNQYPJQDRXPH-UHFFFAOYSA-N 2-cyanobenzohydrazide Chemical compound NNC(=O)C1=CC=CC=C1C#N TWJNQYPJQDRXPH-UHFFFAOYSA-N 0.000 description 1
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 description 1
- 102000053723 Angiotensin-converting enzyme 2 Human genes 0.000 description 1
- 108090000975 Angiotensin-converting enzyme 2 Proteins 0.000 description 1
- 238000009010 Bradford assay Methods 0.000 description 1
- 102100021935 C-C motif chemokine 26 Human genes 0.000 description 1
- 108010059892 Cellulase Proteins 0.000 description 1
- 108010084185 Cellulases Proteins 0.000 description 1
- 102000005575 Cellulases Human genes 0.000 description 1
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- 108091035707 Consensus sequence Proteins 0.000 description 1
- 206010011409 Cross infection Diseases 0.000 description 1
- AEMOLEFTQBMNLQ-YMDCURPLSA-N D-galactopyranuronic acid Chemical compound OC1O[C@H](C(O)=O)[C@H](O)[C@H](O)[C@H]1O AEMOLEFTQBMNLQ-YMDCURPLSA-N 0.000 description 1
- 108700034637 EC 3.2.-.- Proteins 0.000 description 1
- 102100038132 Endogenous retrovirus group K member 6 Pro protein Human genes 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 101150041602 GTF1 gene Proteins 0.000 description 1
- IAJILQKETJEXLJ-UHFFFAOYSA-N Galacturonsaeure Natural products O=CC(O)C(O)C(O)C(O)C(O)=O IAJILQKETJEXLJ-UHFFFAOYSA-N 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 101000897493 Homo sapiens C-C motif chemokine 26 Proteins 0.000 description 1
- 101001093748 Homo sapiens Phosphatidylinositol N-acetylglucosaminyltransferase subunit P Proteins 0.000 description 1
- 239000000232 Lipid Bilayer Substances 0.000 description 1
- 239000006142 Luria-Bertani Agar Substances 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 102000018697 Membrane Proteins Human genes 0.000 description 1
- 101100476480 Mus musculus S100a8 gene Proteins 0.000 description 1
- TUNFSRHWOTWDNC-UHFFFAOYSA-N Myristic acid Natural products CCCCCCCCCCCCCC(O)=O TUNFSRHWOTWDNC-UHFFFAOYSA-N 0.000 description 1
- 235000021360 Myristic acid Nutrition 0.000 description 1
- 239000000020 Nitrocellulose Substances 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- 102100035188 Phosphatidylinositol N-acetylglucosaminyltransferase subunit P Human genes 0.000 description 1
- 241000425347 Phyla <beetle> Species 0.000 description 1
- 239000004743 Polypropylene Substances 0.000 description 1
- 102000029301 Protein S Human genes 0.000 description 1
- 108010066124 Protein S Proteins 0.000 description 1
- 108010001267 Protein Subunits Proteins 0.000 description 1
- 102000002067 Protein Subunits Human genes 0.000 description 1
- 238000002123 RNA extraction Methods 0.000 description 1
- 241000315672 SARS coronavirus Species 0.000 description 1
- 241000008910 Severe acute respiratory syndrome-related coronavirus Species 0.000 description 1
- 239000005708 Sodium hypochlorite Substances 0.000 description 1
- 101710172711 Structural protein Proteins 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 239000012190 activator Substances 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
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000002421 anti-septic effect Effects 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 239000004599 antimicrobial Substances 0.000 description 1
- 229940121357 antivirals Drugs 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 229960000686 benzalkonium chloride Drugs 0.000 description 1
- CADWTSSKOVRVJC-UHFFFAOYSA-N benzyl(dimethyl)azanium;chloride Chemical compound [Cl-].C[NH+](C)CC1=CC=CC=C1 CADWTSSKOVRVJC-UHFFFAOYSA-N 0.000 description 1
- 238000005842 biochemical reaction Methods 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 238000010170 biological method Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- UDSAIICHUKSCKT-UHFFFAOYSA-N bromophenol blue Chemical compound C1=C(Br)C(O)=C(Br)C=C1C1(C=2C=C(Br)C(O)=C(Br)C=2)C2=CC=CC=C2S(=O)(=O)O1 UDSAIICHUKSCKT-UHFFFAOYSA-N 0.000 description 1
- 210000000234 capsid Anatomy 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 229940106157 cellulase Drugs 0.000 description 1
- 239000013626 chemical specie Substances 0.000 description 1
- 239000000460 chlorine Substances 0.000 description 1
- 229910052801 chlorine Inorganic materials 0.000 description 1
- 238000004040 coloring Methods 0.000 description 1
- 238000005094 computer simulation Methods 0.000 description 1
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 239000000287 crude extract Substances 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000001086 cytosolic effect Effects 0.000 description 1
- 230000007123 defense Effects 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 229910000397 disodium phosphate Inorganic materials 0.000 description 1
- 235000019800 disodium phosphate Nutrition 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 239000012149 elution buffer Substances 0.000 description 1
- 230000026502 entry into host cell Effects 0.000 description 1
- 230000006862 enzymatic digestion Effects 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 230000004345 fruit ripening Effects 0.000 description 1
- 230000013595 glycosylation Effects 0.000 description 1
- 101150031572 gtfA gene Proteins 0.000 description 1
- XLYOFNOQVPJJNP-ZSJDYOACSA-N heavy water Substances [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 description 1
- 125000000623 heterocyclic group Chemical group 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 125000000487 histidyl group Chemical group [H]N([H])C(C(=O)O*)C([H])([H])C1=C([H])N([H])C([H])=N1 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 238000007654 immersion Methods 0.000 description 1
- 238000003119 immunoblot Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 150000002614 leucines Chemical class 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 230000002934 lysing effect Effects 0.000 description 1
- 230000002101 lytic effect Effects 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 238000007726 management method Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 229940126601 medicinal product Drugs 0.000 description 1
- 102000006240 membrane receptors Human genes 0.000 description 1
- 108020004084 membrane receptors Proteins 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 235000019799 monosodium phosphate Nutrition 0.000 description 1
- 210000004400 mucous membrane Anatomy 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 229920001220 nitrocellulos Polymers 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
- 239000000590 phytopharmaceutical Substances 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- -1 polypropylene Polymers 0.000 description 1
- 229920001155 polypropylene Polymers 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000006916 protein interaction Effects 0.000 description 1
- 230000004850 protein–protein interaction Effects 0.000 description 1
- 238000006479 redox reaction Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 239000006152 selective media Substances 0.000 description 1
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 230000009897 systematic effect Effects 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/005—Enzyme inhibitors
Definitions
- the present invention relates to the use of a synthetic protein having a high antiviral activity, in particular having the capability of lysis of the SARS-CoV-2 Spike proteins .
- the protein obj ect of the invention can be used in various fields :
- the protein obj ect of the present invention can be used in the preparation of disinfectant solutions that can be used in the outpatient , hospital and domestic field, as a disinfectant for skin and surfaces in environments with high traf fic, in the filters of the air inlet and extraction systems or air conditioners or AHU treatment units , in environments used for the preparation of food .
- the protein obj ect of the present invention can be used in the preparation of disinfectant solutions that can be used in the outpatient , hospital and domestic field, as a disinfectant for skin and surfaces in environments with high traf fic, in the filters of the air inlet and extraction systems or air conditioners or AHU treatment units , in environments used for the preparation of food .
- the filter of the air inlet and extraction systems or air conditioners or AHU treatment units in environments used for the preparation of food .
- In general in all those areas where it is necessary to adopt antimicrobial prophylaxis .
- This biochemical action turns out to be a common character that requires the action of a single conserved mechanism present in the plant world by a class of enzymes : the polygalacturonases that are able to split the bond between the two heterocyclic rings of cellulose , through an endoglucosidase ( cellulase ) typical of bacteria and fungi with a lytic action against the infested plant tissue .
- the fusion of the biological structures that allows the transmission of the genetic material of the virus to the eukaryotic cell occurs through the combination of the Spikes by means of the membrane receptors allowing the introduction of the genetic material . Both of these mechanisms of infection require interaction with the di f ferent characteristics of the surface proteins that allow the pathogens to enter the vegetal eukaryotic or plant cells .
- Polygalacturonases are cellulases , they are proteins belonging to the class of glycosylases , which catalyze the hydrolysis reaction : Polygalacturonic acid + H 2 O Polygalacturonic acid (broken) + Galacturonic acids .
- PG plays an essential role in the fruit ripening process .
- the protopectins are degraded to pectic acids by the action of pectinesterase .
- the pectic acids which are polymers of galacturonic acid, are hydrolyzed and made soluble by PG, with consequent softening of the pulp .
- Plant organisms have developed a defense system based on polygalacturonase inhibiting proteins (PGIPs ) capable of inhibiting the pectinesterase reaction ( Jaillon 0, et al . Nature , 2007 Sep 27 . PMID 17721507 .
- PGIPs polygalacturonase inhibiting proteins
- PGIP PGIP
- a degrading activity composed of a series of repeated leucines that form the so-called " leucine zipper” or leucine zipper that recognizes pectinesterases and degrades them by releasing hydrogen peroxide on contact.
- the ionic species released (H+ + O 2 2 -) , come into close contact with the structures external to the complex and begin to oxidize and reduce by transfer and acquisition of the electrons of the outer layer of the atoms, making up the glycoprotein, coming into contact with the two aforementioned ionic species .
- rich in leucine (LRR) consist of 2-45 motifs of 20-30 amino acids in length that generally fold into an arc or horseshoe shape [1] .
- roteins andcome from eukaryotic viruses which appear to provide a structural framework for the formation of protein-protein interactions [2, 3] .
- the analyzed sequences of the LRRprotein group suggested the existence of different LRRs generating many subfamilies.
- the significance of this classification is that the repetitions of different subfamilies never occur simultaneously and most likely evolved independently, among the plant Phyla.
- LRRprotein families characterized by different lengths and repeated consensus sequences have been identified. Eleven segments of LRR residues (LxxLxLxxN / CxL) , corresponding to the beta strand with the adjacent ring regions, are conserved in the LRRproteins, while the remaining parts of the repeats (defined here as variables) can be very different.
- Some classes of PGIP take shape from a linear sequence of amino acids followed by two halves in "loops" to form a horseshoe-like protein.
- the concave face and adjacent loops are the mostcommon protein interaction surfaces on LRRp roteins.
- the 3D structure of some protein-ligand LRR complexes shows that the concave surface of the LRR domain is ideal for alpha-helix interaction, thus supporting earlier conclusions that the elongated and curved LRR structure provides an outstanding framework for achieving diverse interactions protein-protein [ 2 ] .
- cytoplasmic or anchored to the bacterial membrane The subfamily of PGIP named above with defensive functions is divided into two classes : cytoplasmic or anchored to the bacterial membrane .
- the latter has a portion of myristic acid which allows it to anchor to cell membranes and thus oriented it will go directly into contact with the exogenous glucanic and / or peptido-glucanic structures typical of fungal bacteria and viruses . This contact triggers a redox reaction breaking the bonds of these structures destroying the infesting microorganism [ 6 ] .
- SARS CoV-2 is in particular a virus of the SARS-related coronavirus / SARS-CoV species , belonging to the coronavirus family and having a viral genome consisting of a single RNA helix of about 30 kb .
- the virion has four structural proteins , known as : protein S ( spike ) , E ( envelope ) , M (membrane ) and N (nucleocapsid) ;
- SARS-CoV-2 spike proteins are glycoproteins responsible for coronavirus entry into host cells and consist of two functional subunits , S I and S2 subunits .
- the S I subunit consists of the N-terminal domain (NTD) and the receptor binding domain (RBD) .
- the function of the S I subunit is to bind to the receptor on the host cell .
- the function of the S2 subunit is to fuse the membranes of viruses and host cells .
- the cleavage site at the boundary between S I and S2 subunits is called the S I / S2 cleavage site for proteases .
- This glyco-protein complex begins fusion with cell receptors and then establishes a fusion bond which ultimately results in the transmission of the genetic material within the eukaryotic cell and the consequent destruction of the capsid and the retro-transcription of the virus.
- any activity that requires a high density of people with a possibility of zonal contagion must be subjected again to systematic and also systemic disinfection if it has a hydraulic air distribution system available with devices that must convey by treating thousands of cubic meters per hour .
- the subject of the present invention is a biological method for countering the attack and proliferation of viruses such as SARS-Cov-2 ( coronaviridae ) .
- the obj ect of the invention is in fact the use of a synthetic protein made using gene sequences coding for the polygalacturonase inhibitor derived from vi ti s vinifera to combat viruses in general and SARS-CoV-2 in particular alone or in combination with other drugs , antivirals suitable for the purpose .
- the obj ect of the present invention is a synthetic protein encoded by the sequence having SEQ ID no : 1 and having amino acid sequence of SEQ ID no : 2 and / or a protein having at least 95% sequence identity with SEQ ID no : 2 for the ' ' use in the medical field alone or as a base of antiviral preparations for topical , cutaneous or mucosal use , via aerosol .
- the use of the synthetic protein encoded by the sequence having SEQ ID no : 1 and having amino acid sequence of SEQ ID no : 2 and / or of a protein having at least 95% sequence identity with SEQ ID is also an obj ect of the present invention no : 2 as a disinfectant for environments , skin and surfaces .
- the synthetic protein obj ect of the present invention can be used in the preparation of disinfectant solutions that can be used in the outpatient , hospital and domestic field, on skin and surfaces in high traf fic environments , in filters o f air inlet and extraction systems or air conditioners or of AHU treatment units , in environments used for food preparation . In general , in all those areas where it is necessary to adopt antiviral prophylaxis .
- the present invention also relates to a method for controlling or eliminating viruses , preferably Sars-CoV-2 from environments or surfaces where this method includes the application on the surface or parts thereof of the protein according to the invention or of compositions that include it .
- the present invention also relates to a process for the destruction of glycoproteins included in viruses , preferably Sars-CoV-2 .
- the present invention also relates to a composition comprising said protein at different concentrations which constitutes an antimicrobial solution that can be used in various fields in environmental disinfection.
- Figure 1 Western blot analysis performed on Spike proteins incubated with PGIP and MgC12 for 48 hours. An antibody that recognizes two bands (180 and 80 KDa) with Spike SI + S2 was used for the experiments.
- the object of the present invention is the use in the medical field of a synthetic protein, called PGIP, encoded by the sequence having SEQ ID no: 1 and having amino acid sequence of SEQ ID no: 2 made using a gene sequence coding for the polygalacturonase inhibitor derived from vitis vinifera.
- PGIP synthetic protein
- sequences according to the invention are in any case reported in the attached sequence listing .
- the obj ect of the present invention is therefore the use in the medical field of a protein encoded by the nucleotide sequence of SEQ ID no . 1 and / or a sequence having at least 95% sequence identity with SEQ ID no . 1 ;
- the present invention also relates to the use in the medical field of a protein having the amino acid sequence of SEQ ID no: 2 and / or a sequence having at least 95% sequence identity with SEQ ID no: 2.
- sequence having SEQ ID No: 1 was cloned into a vector for expression in bacteria or yeasts .
- vectors suitable for use according to the invention are known to those skilled in the art, in a preferred embodiment pBE-s DNA are preferably used, and secondly the vector pGAPZa- A (respectively sold by Takara and Thermofisher) .
- the protein according to the invention can be produced in various bacteria and yeasts suitable for the purpose and known to the skilled in the art, in a preferred embodiment the protein according to the invention is produced in e.Coli STELLAR (E. Coli HST08 takara ) or e.Coli DH5a, Pi chi a Pastoris (Gregg et al., 1985; Gregg et al., 1989, Clare et al., 1991a; Clare et al., 1991b; Romanos et al., 1991) and during the transcription of the protein glycosylation of the same brings it into its natural form or hyper-glycosylation increases its metabolic capacity.
- Pichia pastoris the protein is post- translationally hyper-glycosylated in 5 points, its molecular weight varies from 56KDa to 160KDa. This hyper-glycosylation can be removed during purification procedures.
- a tag sequence of 6-10 histidine residues is added to the sequence of the protein according to the invention.
- Other types of tags known to those skilled in the art are however suitable for the purpose according to the invention .
- RT-PCR for the amplification of the gene of SEQ ID no: 1, preferably performed using modified primers comprising sequences recognized by restriction enzymes (Gibson method) so as to provide the amplified with the desired sequence for subsequent digestion;
- the PCR product is subjected to purification, digestion by insertion into a vector, preferably pBE-s DNA, and secondarily into the vector pGAPZa-A, expressly selected for its hyper-expression capacity exclusively in bacteria and yeasts with non-protease activity.
- a vector preferably pBE-s DNA
- the vectors comprising the sequence of SEQ ID no: 1 are used to transform competent cells suitable for the purpose.
- Competent cells are preferably Pichia Pastoris or E. coll.
- the transformation preferably takes place by electroporation since chemical transformation is not excluded.
- the transformed cells are selected, preferably with antibiotics, and multiplied in a suitable medium known to the skilled in the art.
- LB / YPD is preferably used for E. coll and pichia pastoris respectively.
- the cells are lysed to extract the total proteins and to proceed with the purification of the protein of interest preferably on an affinity column.
- the product obtained from the purification step is subjected to encapsulation in lipids, preferably phospholipids in order to facilitate its diffusion and protection from selfoxidation, thus prolonging its stability, the time of residence on the surfaces and favoring its affinity with viral structures. .
- the product obtained from the purification step or from the encapsulation step is subjected to freeze-drying.
- raw or crude extract in the context of the present invention, by raw or crude extract is meant the cellular lysate subjected to centrifugation and sonication but not to purification on an affinity column.
- the engineering of pichia pastoris and E. coll was achieved by electroporation or by transformation of the bacteria competent to accept the plasmid (chemical method) .
- said PGIP coding sequence is cloned in 5'- 3 '"IN FRAME" with the expression structure of the plasmids according to the invention.
- the competent bacteria transformed with the vectors comprising the expression cassette of SEQ ID no. 1 capable of producing the protein according to the invention and labeled with histidine tags were lysed after the adequate culture time. Following the mechanical lysis which took place by sonication of 5 min 'at a frequency> 20kH, on ice, the lysate was passed into the imidazole gradient purification column up to lOOmM to detach, after a first elution, the protein complex . Different samples were run on SD-PAGE gels to verify and demonstrate that the synthetic protein exists, is well characterized and is usable from now on .
- FIG. 1 In particular, in order to verify the effectiveness of the protein according to the invention in fighting viruses, an experiment was set up (fig. 1) , in which the protein according to the invention was placed in contact with the spike protein produced by SARS-CoV-2.
- the SARS-CoV-2 virions are in fact surrounded by a lipid bilayer from which the trimers of the spike protein (S) protrude.
- the heavily glycosylated S trimers bind the ACE2 receptor and mediate the entry of virions into target cells.
- S comprises two functional subunits responsible for binding to the host cell receptor (SI subunit) and for fusion of viral and cell membranes (S2 subunits) .
- the protein according to the invention was puri fied, extracted and placed with or without the addition of 1 mM of MgCl 2 (magnesium acts as a co-enzymatic activator ) , in contact with a mixture of sars-Cov2 spike proteins ( ab-cam) in a ratio of 5 : 1 ; the protein according to the invention is present at the final concentration in Img / ml .
- the experiment was carried out with both the protein extracted from and . Coli both with that extracted from P . Pastoris placed in contact with both the S I and S2 portions and with the S I portion only .
- Table 1 shows the values of the densitometric analyzes Table 1 :
- the obj ect of the present invention is therefore a protein encoded by the sequence having SEQ ID No : 1 and / or by a sequence having at least 95% sequence identity with SEQ ID no : 1 for use in the medical field .
- the present invention also relates to a protein having an amino acid sequence of SEQ ID no : 2 and / or a protein having at least 95% sequence identity with SEQ ID no : 2 for use in the medical field .
- Still the subj ect of the present invention is said protein for use as a medicinal product defined by its antiviral function, in particular against Sars-Cov-
- the protein according to the invention can be used in compositions formulated in liquid form, as a cream or lotion or as a gel . or spray for topical or mucosal applications .
- the topical applications themselves include applications on the skin and mucous membranes .
- the carriers can be all those used in the pharmaceutical and cosmetic fields .
- the adj uvants and carriers are those cosmetically and pharmaceutically acceptable , as well as the adj uvants and carriers used in the phytopharmaceutical field .
- Carriers include lipid carriers , preferably single and multi-lamellar liposomes ; in a preferred embodiment , the protein according to the invention is in fact packaged or encapsulated in said structures to allow more ef fective delivery to the treatment site , better dif fusion and protection from sel f-oxidation, thus extending the time of residence on the surfaces , and promoting af finity with viral structures .
- the protein according to the invention is preferably administered topically, cutaneously and / or oropharyngeal- nasal and can be formulated in sprays , aerosols for inhalation, gels , creams and lotions .
- the obj ect of the present invention is therefore a composition comprising the protein having SEQ ID no : 2 and / or a protein having 95% sequence identity with SEQ ID no : 2 ; optionally said composition comprises at least one of saline buf fer, preferably PBS , protease inhibitor, MgC12 , pharmaceutically acceptable excipients , carriers , thickeners and gelling agents .
- the composition according to the invention further comprises cellulose , preferably methylcellulose .
- the composition comprising the protein according to the invention further comprises at least one antiviral drug .
- composition comprising the protein according to the invention can also be formulated in spray, semi-liquid, creamy, semi-solid or solid forms , creams , suspensions , milks or soaps .
- the composition according to the invention can also be composed of a lysate of microorganisms expressing the protein having SEQ ID no : 2 and / or a protein having at least 95% sequence identity with SEQ ID no : 2 ;
- the protein having amino acid sequence of SEQ ID no : 2 and / or amino acid sequence having at least 95% sequence identity with SEQ ID no : 2 can therefore be used as an environmental and skin disinfectant .
- the protein according to the invention can be used alone or included in a composition further comprising vectors known to those skilled in the art and can be applied by spraying, formulated in gel or applied in solution .
- a composition comprising the protein according to the invention can therefore be formulated in spray, semiliquid, semi-solid, solid, suspension, or gel form to be applied on the surfaces to be treated .
- the application can also be a spray .
- compositions comprising the protein according to the invention can be used in household hygiene products as a disinfectant; in skin disinfectants, in soaps etc. ex.
- composition comprising the protein according to the invention can further comprise at least one of saline buffer, preferably PBS, protease inhibitor, MgC12, cellulose and methyl cellulose, gelling agents, preferably methyl orixane or alginates such as calcium or sodium alginate and a lysate of microorganisms, preferably Pichia pastoris or E. coli, expressing the protein itself.
- saline buffer preferably PBS
- protease inhibitor MgC12
- cellulose and methyl cellulose gelling agents
- gelling agents preferably methyl orixane or alginates such as calcium or sodium alginate and a lysate of microorganisms, preferably Pichia pastoris or E. coli, expressing the protein itself.
- the object of the present invention is therefore a method for the control or elimination of viruses, in particular Sars-CoV- 2 from environments or surfaces where this method includes the application on the surface or on parts of it of at least one of
- lysate of microorganisms preferably Pichia pastoris or E. coli, expressing the protein having SEQ ID no 2 and / or a protein having at least 95% sequence identity with SEQ ID no: 2
- compositions comprising said protein.
- the protein according to the invention resists 48-72h on surfaces at ambient T.
- the subject of the present invention is a method for the control or elimination of viruses, preferably Sars-CoV-2 from environments or surfaces where this method includes the application on the surface or parts thereof of at least one of
- a lysate of microorganisms preferably pichia pastoris or E.coli, expressing the protein having SEQ ID no: 2 and / or a protein having at least 95% sequence identity with SEQ ID no: 2
- DNA insert from 10 to 100 ng, molar excess 3: 1 compared to the Vector DNA using T4 DNA HD CLONING Master Mix 5 pL / 20pL Nuclease free water to taste
- the plasmid that includes the gene of interest is replicated to obtain the necessary quantities using DH5a cells according to methods known to the expert in the field .
- Electroporation in e.Coli / Pichia Pastoris 1. Place 1.5 ml tubes containing PIGP competent cells and electro-competent e.coli / pichia pastoris on ice.
- Spike proteins, ready to use are left to react with the purified and isolated protein after having been purified in a 10-200mM imidazole gradient on an IMAC His-tag column.
- the reading of the purified protein concentration reported a spectrometric reading at 595nm, according to the Bradford method, obtaining the concentration of Img /ml.
- the experiment using the protein characterized in Fig. 3 was conducted in such a way as to have a solution of the spike proteins and the protein, according to the invention, in a 1 : 5 ratio. This mix is left to react for Ih, 24h and 48h.
- the gel was then left for 24 hours in the bleaching solution to which 50% methanol-10% acetic acid was added) .
- the samples were run at constant 100V for 70min by placing it on nitrocellulose in running buffer and subjected to 380mA for 90min.
- the membrane was colored with a solution of Ponceau S (Sigma) and then decoloured with double distilled water until the red color disappeared completely.
- the membrane was then incubated in 100 ml of saturation solution consisting of IX PBS (pH 7.2: 80 mM Na2HPO4; 20 mM NaH2PO4 x 2H2O; 100 mM NaCl) ; 0.1% Tween 20; 8 gr of dry milk) for 16-18 hours at 4 ° C. After washing with 0. IX PBS and 0.1% Tween 20 (Sigma) .
- the membrane was incubated with primary mouse anti-spike-tag monoclonal antibody (Ab-Cam) diluted in PBS 1: 5000 at room temperature for 1 hour. It is then washed again washed with the PBS-Tween 20 solution, six times for 5 'min.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Immunology (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Peptides Or Proteins (AREA)
Abstract
A protein encoded by the sequence having SEQ ID No : 1 and / or by a sequence having at least 95% sequence identity with SEQ ID no : 1 for use in the medical field.
Description
SYNTHETIC PROTEIN FOR USE IN THE MEDICAL FIELD
Technical field
The present invention relates to the use of a synthetic protein having a high antiviral activity, in particular having the capability of lysis of the SARS-CoV-2 Spike proteins . The protein obj ect of the invention can be used in various fields :
- in environmental disinfection, the protein obj ect of the present invention can be used in the preparation of disinfectant solutions that can be used in the outpatient , hospital and domestic field, as a disinfectant for skin and surfaces in environments with high traf fic, in the filters of the air inlet and extraction systems or air conditioners or AHU treatment units , in environments used for the preparation of food . In general , in all those areas where it is necessary to adopt antimicrobial prophylaxis .
- in the medical field, alone or as a base for antiviral preparations or to combat infections for topical , cutaneous or mucosal use , via aerosol . As an ingredient in mouthwashes , toothpastes and skin creams .
State of the art
There are many bacteria and many viruses that have in common as an attacking mechanism the target biological structures , which are made up of proteins with a structural function, located inside the plasma membrane in order to give it mechanical resistance . At the end of the interaction of the biochemical reaction between the two species , a permeation is obtained which ultimately leads to the destruction of the cell wall , through lysis . This biochemical action turns out to be a common character that requires the action of a single conserved mechanism present in the plant world by a class of
enzymes : the polygalacturonases that are able to split the bond
between the two heterocyclic rings of cellulose , through an endoglucosidase ( cellulase ) typical of bacteria and fungi with a lytic action against the infested plant tissue . In other cases , however, especially with regard to virus attacks , the fusion of the biological structures that allows the transmission of the genetic material of the virus to the eukaryotic cell occurs through the combination of the Spikes by means of the membrane receptors allowing the introduction of the genetic material . Both of these mechanisms of infection require interaction with the di f ferent characteristics of the surface proteins that allow the pathogens to enter the vegetal eukaryotic or plant cells .
Polygalacturonases (PG) are cellulases , they are proteins belonging to the class of glycosylases , which catalyze the hydrolysis reaction : Polygalacturonic acid + H2O Polygalacturonic acid (broken) + Galacturonic acids . PG plays an essential role in the fruit ripening process . During maturation the protopectins are degraded to pectic acids by the action of pectinesterase . Subsequently, the pectic acids , which are polymers of galacturonic acid, are hydrolyzed and made soluble by PG, with consequent softening of the pulp . Plant organisms have developed a defense system based on polygalacturonase inhibiting proteins ( PGIPs ) capable of inhibiting the pectinesterase reaction ( Jaillon 0, et al . Nature , 2007 Sep 27 . PMID 17721507 .
Among the various classes of PGIP , there is one with a degrading activity, composed of a series of repeated leucines that form the so-called " leucine zipper" or leucine zipper
that recognizes pectinesterases and degrades them by releasing hydrogen peroxide on contact. The ionic species released (H+ + O2 2-) , come into close contact with the structures external to the complex and begin to oxidize and reduce by transfer and acquisition of the electrons of the outer layer of the atoms, making up the glycoprotein, coming into contact with the two aforementioned ionic species . rich in leucine (LRR) consist of 2-45 motifs of 20-30 amino acids in length that generally fold into an arc or horseshoe shape [1] . roteins andcome from eukaryotic viruses, which appear to provide a structural framework for the formation of protein-protein interactions [2, 3] .
The analyzed sequences of the LRRprotein group suggested the existence of different LRRs generating many subfamilies. The significance of this classification is that the repetitions of different subfamilies never occur simultaneously and most likely evolved independently, among the plant Phyla.
However, it is now clear that all major LRR classes had curved horseshoe-shaped structures with a parallel beta sheet on the concave side and mainly helical elements on the convex side. At least six LRRprotein families characterized by different lengths and repeated consensus sequences have been identified. Eleven segments of LRR residues (LxxLxLxxN / CxL) , corresponding to the beta strand with the adjacent ring regions, are conserved in the LRRproteins, while the remaining parts of the repeats (defined here as variables) can be very different. Some classes of PGIP take shape from a linear sequence of amino acids followed by two halves in "loops" to form a horseshoe-like protein.
The concave face and adjacent loops are the mostcommon protein interaction surfaces on LRRp roteins. The 3D structure of some protein-ligand LRR complexes shows that the concave surface of the LRR domain is ideal for alpha-helix interaction, thus
supporting earlier conclusions that the elongated and curved LRR structure provides an outstanding framework for achieving diverse interactions protein-protein [ 2 ] .
The prediction of the molecular computational model suggests that the conserved model LxxLxL, which is shorter than the previously proposed LxxLxLxxN / CxL is suf ficient to impart the characteristic horseshoe , with protein curvature withrepeats of 20 to 30 residues [ 5 ] .
The subfamily of PGIP named above with defensive functions is divided into two classes : cytoplasmic or anchored to the bacterial membrane . The latter has a portion of myristic acid which allows it to anchor to cell membranes and thus oriented it will go directly into contact with the exogenous glucanic and / or peptido-glucanic structures typical of fungal bacteria and viruses . This contact triggers a redox reaction breaking the bonds of these structures destroying the infesting microorganism [ 6 ] .
SARS CoV-2 is in particular a virus of the SARS-related coronavirus / SARS-CoV species , belonging to the coronavirus family and having a viral genome consisting of a single RNA helix of about 30 kb . The virion has four structural proteins , known as : protein S ( spike ) , E ( envelope ) , M (membrane ) and N (nucleocapsid) ; SARS-CoV-2 spike proteins are glycoproteins responsible for coronavirus entry into host cells and consist of two functional subunits , S I and S2 subunits . The S I subunit consists of the N-terminal domain (NTD) and the receptor binding domain (RBD) . The function of the S I subunit is to bind to the receptor on the host cell . The function of the S2 subunit is to fuse the membranes of viruses and host cells . The cleavage site at the boundary between S I and S2 subunits is called the S I / S2 cleavage site for proteases .
This glyco-protein complex begins fusion with cell receptors and then establishes a fusion bond which ultimately results in the transmission of the genetic material within the eukaryotic
cell and the consequent destruction of the capsid and the retro-transcription of the virus.
Given the economic importance, numerous products have been developed over the years that are able to counteract the attack of the aforementioned microorganisms.
There is a strong need for new methods to combat these types of infections that are increasingly problematic for management, the danger of transmissibility. The current use of antiseptic products capable of environmentally containing the spread of viral loads is currently borne by very aggressive chemicals such as: Na+ HCLO-(sodium hypochlorite) , Benzalkonium chloride, or through glutarladehyde or substances such as , some species of acids that contain an active free chlorine activity between 0.1% and 0.5%. These chemical species with redox activity, however, have a short life, since by combining with atmospheric oxygen O2 they become inert quickly. For example with hypochlorite :
the inertization reaction is the above.
The reaction that occurs shortly after if there are no biological structures to be reduced, and therefore the disinfection process in various capacities, including spraying of various types of surfaces, must be repeated.
In the same way, any activity that requires a high density of people with a possibility of zonal contagion, must be subjected again to systematic and also systemic disinfection if it has a hydraulic air distribution system available with devices that must convey by treating thousands of cubic meters per hour .
Summary of the invention
The subject of the present invention is a biological method for countering the attack and proliferation of viruses such as SARS-Cov-2 ( coronaviridae ) .
The obj ect of the invention is in fact the use of a synthetic protein made using gene sequences coding for the polygalacturonase inhibitor derived from vi ti s vinifera to combat viruses in general and SARS-CoV-2 in particular alone or in combination with other drugs , antivirals suitable for the purpose .
The obj ect of the present invention is a synthetic protein encoded by the sequence having SEQ ID no : 1 and having amino acid sequence of SEQ ID no : 2 and / or a protein having at least 95% sequence identity with SEQ ID no : 2 for the ' ' use in the medical field alone or as a base of antiviral preparations for topical , cutaneous or mucosal use , via aerosol . As an ingredient in mouthwashes , toothpastes and skin creams .
The use of the synthetic protein encoded by the sequence having SEQ ID no : 1 and having amino acid sequence of SEQ ID no : 2 and / or of a protein having at least 95% sequence identity with SEQ ID is also an obj ect of the present invention no : 2 as a disinfectant for environments , skin and surfaces . The synthetic protein obj ect of the present invention can be used in the preparation of disinfectant solutions that can be used in the outpatient , hospital and domestic field, on skin and surfaces in high traf fic environments , in filters o f air inlet and extraction systems or air conditioners or of AHU treatment units , in environments used for food preparation . In general , in all those areas where it is necessary to adopt antiviral prophylaxis .
The present invention also relates to a method for controlling or eliminating viruses , preferably Sars-CoV-2 from environments or surfaces where this method includes the application on the surface or parts thereof of the protein according to the invention or of compositions that include it . The present invention also relates to a process for the destruction of glycoproteins included in viruses , preferably Sars-CoV-2 .
The present invention also relates to a composition comprising said protein at different concentrations which constitutes an antimicrobial solution that can be used in various fields in environmental disinfection.
Further objects and advantages will become apparent from the detailed description of the invention.
Description of the figures
Figure 1: Western blot analysis performed on Spike proteins incubated with PGIP and MgC12 for 48 hours. An antibody that recognizes two bands (180 and 80 KDa) with Spike SI + S2 was used for the experiments.
Panel A - Results on SARS-CoV2 Spike SI + S2. Wells: 1st spike SI S2 - L ladder - 2nd PGIP E.coli -3a spike SI S2 + MgC12 - 4th PGIP P. pastoris
Panel B - Results on SARS-CoV2 Spike SI. Wells: lb spike SI - L ladder - 2b PGIP E.coli - 3b spike SI + MgC12 - 4b PGIP P. pastoris
Detailed description of the invention:
The object of the present invention is the use in the medical field of a synthetic protein, called PGIP, encoded by the sequence having SEQ ID no: 1 and having amino acid sequence of SEQ ID no: 2 made using a gene sequence coding for the polygalacturonase inhibitor derived from vitis vinifera.
The sequence was selected after several pairing studies, in particular as described in WO2019077477.
SEQ ID NO:1 - sequence of PGIP, polygalacturanase inhibiting protein from vitis vinifera. gagtctggtggagaattcgaattcgaattcatggagacttcaaaactttttcttctctcct cctctctcctcctagtcttactcgccactcgtccatgtccttctctctctgaacgttgcaa
cccaaaagacaaaaaagttctccttcaaatcaaaaaagccctagacaatccctacattcta gcttcgtggaatcccaacaccgattgctgcggatggtactgcgtcgaatgtgacct cacca cccaccgcatcaactcgctcaccatcttctccggccagctatccggccagattcccgacgc tgttggtgaccttccgttcctcgagaccctcatcttccgcaagctctctaacctcaccggt caga t cccgccggcgattgccaaactcaagcacctaaaaatggtt cgcc tt agc tggacca acct ctccggtcccgtgccggcgttcttcagcgagcttaagaacctcacgt acct cgacct ctccttcaataacctatctggacccattcccggcagcctctctctcctccccaacctcggc gcactccatctcgaccggaaccacctcacaggcccaatccctgactccttcggaaaattcg ccggctctaccccaggtctacacctctcacacaaccaactttccgggaaaatcccatattc tttcagaggattcgaccccaatgtcatggacttatcgcgtaacaagcttgagggtgacctg tcaatattcttcaatgccaataagtcaacacagatcgttgacttctcacggaacttgttcc agtttgatctttcgagagtggaattcccgaagagtttgacgtcgttggacctttcgcataa caagatcgccgggagcctgccggagatgatgacttctctggatttacagttcctgaacgtg agttacaatcgtttgtgtggtaagattccggtgggtgggaagttgcagagcttcgattacg actcctactttcacaatcggtgcttgtgtggtgctccactccagagctgcaagggtggtgg tggttctggtggtggtggttctggtggtggtggttctgagtctggtggaagttcttttgat tttatggatggttatgataagcctgtgaaagggagaaaaatcaattggatgaaagccggca tattagaatcagacagg
SEQ ID NO : 2 - Sequenza amminoacidica della proteina secondo 1 ' invenzione
ELCNPQDKQALLQIKKDLGNPTTLSSWLPTTDCCNRTWLGVLCDTDTQTYRVNNLDLSGLN LPKPYPIPSSLANLPYLNFLYIGGINNLVGPIPPAIAKLTQLHYLYITHTNVSGAIPDFLS QIKTLVTLDFSYNALSGTLPPS ISSLPNLVGITFDGNRISGAIPDSYGSFSKLFTSMTISR NRLTGKIPPTFANLNLAFVDLSRNMLEGDASVLFGSDKNTQKIHLAKNSLAFDLGKVGLSK NLNGLDLRNNRIYGTLPQGLTQLKFLHSLNVSFNNLCGEIPQGGNLQRFDVSAYANNKCLC GSPLPAC
The sequences according to the invention are in any case reported in the attached sequence listing .
The obj ect of the present invention is therefore the use in the medical field of a protein encoded by the nucleotide sequence of SEQ ID no . 1 and / or a sequence having at least 95% sequence identity with SEQ ID no . 1 ;
The present invention also relates to the use in the medical field of a protein having the amino acid sequence of SEQ ID no: 2 and / or a sequence having at least 95% sequence identity with SEQ ID no: 2.
In order to obtain the expression and to be able to purify the protein according to the invention, the sequence having SEQ ID No: 1 was cloned into a vector for expression in bacteria or yeasts .
The vectors suitable for use according to the invention are known to those skilled in the art, in a preferred embodiment pBE-s DNA are preferably used, and secondly the vector pGAPZa- A (respectively sold by Takara and Thermofisher) .
The protein according to the invention can be produced in various bacteria and yeasts suitable for the purpose and known to the skilled in the art, in a preferred embodiment the protein according to the invention is produced in e.Coli STELLAR (E. Coli HST08 takara ) or e.Coli DH5a, Pi chi a Pastoris (Gregg et al., 1985; Gregg et al., 1989, Clare et al., 1991a; Clare et al., 1991b; Romanos et al., 1991) and during the transcription of the protein glycosylation of the same brings it into its natural form or hyper-glycosylation increases its metabolic capacity. In Pichia pastoris the protein is post- translationally hyper-glycosylated in 5 points, its molecular weight varies from 56KDa to 160KDa. This hyper-glycosylation can be removed during purification procedures.
In one embodiment, a tag sequence of 6-10 histidine residues (His-Tag) is added to the sequence of the protein according to the invention. Other types of tags known to those skilled in the art are however suitable for the purpose according to the invention .
To obtain the protein according to the invention, the method applied was the following:
1) RNA extraction from vitis vinifera
2) RT-PCR for the amplification of the gene of SEQ ID no: 1, preferably performed using modified primers comprising
sequences recognized by restriction enzymes (Gibson method) so as to provide the amplified with the desired sequence for subsequent digestion;
3) Following amplification, the PCR product is subjected to purification, digestion by insertion into a vector, preferably pBE-s DNA, and secondarily into the vector pGAPZa-A, expressly selected for its hyper-expression capacity exclusively in bacteria and yeasts with non-protease activity.
5) The vectors comprising the sequence of SEQ ID no: 1 are used to transform competent cells suitable for the purpose. Competent cells are preferably Pichia Pastoris or E. coll. The transformation preferably takes place by electroporation since chemical transformation is not excluded.
6) The transformed cells are selected, preferably with antibiotics, and multiplied in a suitable medium known to the skilled in the art. LB / YPD is preferably used for E. coll and pichia pastoris respectively.
7) After an adequate culture time selected on the basis of the microorganism, preferably 24h-48h, the cells are lysed to extract the total proteins and to proceed with the purification of the protein of interest preferably on an affinity column. Optionally, the product obtained from the purification step is subjected to encapsulation in lipids, preferably phospholipids in order to facilitate its diffusion and protection from selfoxidation, thus prolonging its stability, the time of residence on the surfaces and favoring its affinity with viral structures. .
Optionally, the product obtained from the purification step or from the encapsulation step is subjected to freeze-drying.
In the context of the present invention, by raw or crude extract is meant the cellular lysate subjected to centrifugation and sonication but not to purification on an affinity column.
In one embodiment, the engineering of pichia pastoris and E. coll was achieved by electroporation or by transformation of the bacteria competent to accept the plasmid (chemical method) . In one embodiment, said PGIP coding sequence is cloned in 5'- 3 '"IN FRAME" with the expression structure of the plasmids according to the invention.
In order to verify the correct production of the protein according to the invention, the competent bacteria transformed with the vectors comprising the expression cassette of SEQ ID no. 1 capable of producing the protein according to the invention and labeled with histidine tags, were lysed after the adequate culture time. Following the mechanical lysis which took place by sonication of 5 min 'at a frequency> 20kH, on ice, the lysate was passed into the imidazole gradient purification column up to lOOmM to detach, after a first elution, the protein complex . Different samples were run on SD-PAGE gels to verify and demonstrate that the synthetic protein exists, is well characterized and is usable from now on .
In order to verify the efficacy of the protein according to the invention in countering or destroying Sars-Cov2, experiments were set up, in which the protein under various conditions was placed in contact with the spike protein produced by Sars Cov-2.
In particular, in order to verify the effectiveness of the protein according to the invention in fighting viruses, an experiment was set up (fig. 1) , in which the protein according to the invention was placed in contact with the spike protein produced by SARS-CoV-2. The SARS-CoV-2 virions are in fact surrounded by a lipid bilayer from which the trimers of the spike protein (S) protrude. The heavily glycosylated S trimers bind the ACE2 receptor and mediate the entry of virions into target cells. S comprises two functional subunits responsible for binding to the host cell receptor (SI subunit) and for fusion of viral and cell membranes (S2 subunits) .
The protein according to the invention was puri fied, extracted and placed with or without the addition of 1 mM of MgCl2 (magnesium acts as a co-enzymatic activator ) , in contact with a mixture of sars-Cov2 spike proteins ( ab-cam) in a ratio of 5 : 1 ; the protein according to the invention is present at the final concentration in Img / ml . The experiment was carried out with both the protein extracted from and . Coli both with that extracted from P . Pastoris placed in contact with both the S I and S2 portions and with the S I portion only .
The result of this contact was observed after 48 hours , with signi ficant ef fects . Following the experiment , the samples were run on SDS-PAGE gel and in figure 1 are represented the membranes that after the blot were incubated with anti-spike antibodies . In panels A and B and in the table below, the result of the contact experiments is represented both with the portions S I and S2 , and only S I in the presence of ImM magnesium; it is possible to observe in wells 2 and 4 how the protein according to the invention is derived from E . coli and P . pastoris is ef fective in lysing the viral spike proteins leading to a reduction of 20-30% of the same after 48h .
Table 1 shows the values of the densitometric analyzes Table 1 :
Medical field
The obj ect of the present invention is therefore a protein encoded by the sequence having SEQ ID No : 1 and / or by a sequence having at least 95% sequence identity with SEQ ID no : 1 for use in the medical field .
The present invention also relates to a protein having an amino acid sequence of SEQ ID no : 2 and / or a protein having at least 95% sequence identity with SEQ ID no : 2 for use in the medical field . Still the subj ect of the present invention is said protein for use as a medicinal product defined by its antiviral function, in particular against Sars-Cov-
The protein according to the invention can be used in compositions formulated in liquid form, as a cream or lotion or as a gel . or spray for topical or mucosal applications . The topical applications themselves include applications on the skin and mucous membranes . The carriers can be all those used in the pharmaceutical and cosmetic fields . The adj uvants and carriers are those cosmetically and pharmaceutically acceptable , as well as the adj uvants and carriers used in the phytopharmaceutical field . Carriers include lipid carriers , preferably single and multi-lamellar liposomes ; in a preferred embodiment , the protein according to the invention is in fact packaged or encapsulated in said structures to allow more ef fective delivery to the treatment site , better dif fusion and protection from sel f-oxidation, thus extending the time of residence on the surfaces , and promoting af finity with viral structures .
The protein according to the invention is preferably administered topically, cutaneously and / or oropharyngeal- nasal and can be formulated in sprays , aerosols for inhalation, gels , creams and lotions . The obj ect of the present invention is therefore a composition comprising the protein having SEQ ID no : 2 and / or a protein having 95% sequence identity with SEQ ID no : 2 ; optionally said composition comprises at least one of saline buf fer, preferably PBS , protease inhibitor, MgC12 , pharmaceutically acceptable excipients , carriers , thickeners and gelling agents . In a preferred embodiment , the composition according to the invention further comprises cellulose , preferably methylcellulose .
In a preferred embodiment , the composition comprising the protein according to the invention further comprises at least one antiviral drug .
The composition comprising the protein according to the invention can also be formulated in spray, semi-liquid, creamy, semi-solid or solid forms , creams , suspensions , milks or soaps . The composition according to the invention can also be composed of a lysate of microorganisms expressing the protein having SEQ ID no : 2 and / or a protein having at least 95% sequence identity with SEQ ID no : 2 ;
Environmental disinfectant
The results of the in vitro experimentation shown in the figure have shown a remarkable activity of the protein according to the invention against the glycoproteins present on the viral surface .
The protein having amino acid sequence of SEQ ID no : 2 and / or amino acid sequence having at least 95% sequence identity with SEQ ID no : 2 can therefore be used as an environmental and skin disinfectant . The protein according to the invention can be used alone or included in a composition further comprising vectors known to those skilled in the art and can be applied by spraying, formulated in gel or applied in solution . A composition comprising the protein according to the invention can therefore be formulated in spray, semiliquid, semi-solid, solid, suspension, or gel form to be applied on the surfaces to be treated . The application can also be a spray .
This composition can also be used as a functional disinfectant base in the field of disinfection in various areas , for example in the outpatient , hospital and domestic field, on skin and surfaces , in high traf fic environments , in filters of inlet and extraction systems , air conditioners or AHU treatment units , in environments used for food preparation . In general , in all those areas where it is necessary to adopt a
disinfectant prophylaxis to counter viral multiplication. By way of non-limiting example, compositions comprising the protein according to the invention can be used in household hygiene products as a disinfectant; in skin disinfectants, in soaps etc. ex. in the disinfection of the intact skin, for example in the disinfection of the hands in the preoperative phase; in hospital wards, against the transmission of nosocomial cross-infections; in disinfectants or community hygiene products (e.g. hotels, airports, schools, doctors' offices or dental offices) ; in the disinfection of surgical instruments .
The composition comprising the protein according to the invention can further comprise at least one of saline buffer, preferably PBS, protease inhibitor, MgC12, cellulose and methyl cellulose, gelling agents, preferably methyl orixane or alginates such as calcium or sodium alginate and a lysate of microorganisms, preferably Pichia pastoris or E. coli, expressing the protein itself.
The object of the present invention is therefore a method for the control or elimination of viruses, in particular Sars-CoV- 2 from environments or surfaces where this method includes the application on the surface or on parts of it of at least one of
- a lysate of microorganisms, preferably Pichia pastoris or E. coli, expressing the protein having SEQ ID no 2 and / or a protein having at least 95% sequence identity with SEQ ID no: 2
- a protein having SEQ ID no: 2 and / or a protein having at least 95% sequence identity with SEQ ID no: 2
- compositions comprising said protein.
From the tests carried out, the protein according to the invention resists 48-72h on surfaces at ambient T.
Finally, the subject of the present invention is a method for the control or elimination of viruses, preferably Sars-CoV-2
from environments or surfaces where this method includes the application on the surface or parts thereof of at least one of
- a lysate of microorganisms, preferably pichia pastoris or E.coli, expressing the protein having SEQ ID no: 2 and / or a protein having at least 95% sequence identity with SEQ ID no: 2
- a protein the protein having SEQ ID no: 2 and / or a protein having at least 95% sequence identity with SEQ ID no: 2
- compositions such as those described above
PROTOCOLS and EXAMPLES:
Cloning of pBE-s DNA and pGAPZ ALPHA A Prl 201-AN:
1) Gently mix fresh competent E. coli STELLAR (takara) cells and transfer 100 pl to a polypropylene tube.
2) Add to the 100 pl of E.coli STELLAR cells in quantities < 10 ng .
3) Incubate in an ice bath for 30 ' .
4) Incubate at + 42 ° C for 43 ' ' .
5) Return to the ice bath for 1-2 ' .
6) Add the SOC medium, pre-incubated at + 37 ° C up to a final volume of 1 ml.
7) Incubate by shaking at 160-225 rpm for 1 hour at + 37 ° C.
9) Plate on selective media, typically less than 100 pl for each 9 cm diameter plate.
10) Incubate overnight at + 37 ° C
11) Selection of the colonies and amplification of the same by incubation overnight at + 37 ° C in LB plate with the selective antibiotic for which the plasmid has the specific resistance kanamycin / ampicillin.
PURIFICATION of the plasmids after cloning
After centrifugation of the liquid, 250 ml of resuspension solution are added to the cell pellet, then 250Lysisof
neutralization solution are mixedmlsolution and 350ml, then centrifuged at 14,000 RPM for 5 min. Subsequently, the content is placed in a purification column and centrifuged at 14,000 RPM for 1 min. Once the eluate has been discarded, 500 mlofwash solution" are added twice. Once the eluate has been discarded, 50 ml of "elution buffer" are placed in the columnandthe concentration of the purity of the plasmid is calculated on the spectrophotometer, which is preserved at - 20 ° C.
Enzymatic cutting and linearization of the vector.
Multiple reaction of enzymatic digestion and linearization. In a final volume of 20 pl, mix: Buffer 10PGIP + GTF1 2 pL ALPHA A Da in a quantity ranging from 0.2 to 1 pg Restriction enzymes as follows: pGAPZ KpNI pL 1 pL XBAI water Nuclease free qb The reaction proceeds according to the protocol known to the expert in the field.
Ligation of the products In a final volume of 20 pl mix: DNA of the linearized vector as above. Similarly linearized PGIP fragment.
DNA insert from 10 to 100 ng, molar excess 3: 1 compared to the Vector DNA using T4 DNA HD CLONING Master Mix 5 pL / 20pL Nuclease free water to taste
Cloning in E.coli / pichia Pastoris
Following ligation, the plasmid that includes the gene of interest is replicated to obtain the necessary quantities using DH5a cells according to methods known to the expert in the field .
Electroporation in e.Coli / Pichia Pastoris
1. Place 1.5 ml tubes containing PIGP competent cells and electro-competent e.coli / pichia pastoris on ice.
2. Add 6 pl (1 ng) of binary vector plasmid DNA to 20 pl of competent cells and mix gently.
3. Place the 0.1 cm electroporation cuvette on ice.
4. set the Gene Pulser II to 25 pF, 200 Ω and 2 - 2.5 kV. * 1
5. Transfer the cells and DNA prepared in step 2 to the electroporation and electroporation cuvette.
6. Remove the cuvette from the electroporator , add 1 mL of SOC * 2 media and transfer to a 14 mL round bottom tube.
7. Incubate for 1 hour at 30 ° C, shaking at 100 rpm.
8. Plate 50 - 100 pl of cells on LB agar plates with 50 pg / ml kanamycin / 10 pg zeocin (second vector) * 3 and incubate for up to 48 hours at 30 ° C.
9. Amplify the colony in liquid LB with Kanamycin / 10 pg zeocin at 30 ° C.
Contact test with spike proteins and relative immunoblot
Spike proteins, ready to use (ab-cam) are left to react with the purified and isolated protein after having been purified in a 10-200mM imidazole gradient on an IMAC His-tag column. The reading of the purified protein concentration reported a spectrometric reading at 595nm, according to the Bradford method, obtaining the concentration of Img /ml. The experiment using the protein characterized in Fig. 3 was conducted in such a way as to have a solution of the spike proteins and the protein, according to the invention, in a 1 : 5 ratio. This mix is left to react for Ih, 24h and 48h. These solutions in volume of 10 ml are then loaded into the wells of a precast gel (pharmacia) in a 5-10% acrylamide gradient, after mixing with 2 pl loading buffer (4% SDS 10% 2-mercaptoethanol 20% glycerol 0.004% bromophenol blue 0.125 M Tris-HCl pH 6.8) while the running buffer consisted of 25 mM Tris 190 mM glycine 0.1% SDS) . The gel was fixed and colored by immersion in the coloring solution (625 mM coomassie brilliant-blue; 50%
methanol; 10% acetic acid) for 30 min., Then, after photographic detection, it is decoloured with the solution (50% methanol-10 % acetic acid) . The gel was then left for 24 hours in the bleaching solution to which 50% methanol-10% acetic acid was added) . After the bleaching, the samples were run at constant 100V for 70min by placing it on nitrocellulose in running buffer and subjected to 380mA for 90min. To verify the outcome of the electro-transfer, the membrane was colored with a solution of Ponceau S (Sigma) and then decoloured with double distilled water until the red color disappeared completely. The membrane was then incubated in 100 ml of saturation solution consisting of IX PBS (pH 7.2: 80 mM Na2HPO4; 20 mM NaH2PO4 x 2H2O; 100 mM NaCl) ; 0.1% Tween 20; 8 gr of dry milk) for 16-18 hours at 4 ° C. After washing with 0. IX PBS and 0.1% Tween 20 (Sigma) . The membrane was incubated with primary mouse anti-spike-tag monoclonal antibody (Ab-Cam) diluted in PBS 1: 5000 at room temperature for 1 hour. It is then washed again washed with the PBS-Tween 20 solution, six times for 5 'min. and incubated with the secondary antibody diluted 1: 10,000 (rabbit anti-mouse conjugated with peroxidase, Sigma) at room temperature for 1 hour. After 6 washes with PBS-Tween 20 the proteins recognized by the antibody with the ECL method (Amersham) were visualized, following the instructions of the supplier company. bibliography
Ausubel, FM, Brent, R., Kingston, RE, Moore, DD, Seidman, JG, Smith, JA, and Struhl, K. (1994) Current
Protocols in Molecular Biology, Greene Publishing Associates and Wiley-Interscience, New York
Baron , M., Reynes, JP, Stassi, D., and Tiraby, G. (1992) A Selectable Bifunctional b-Galactosidase : Phleomycinresistance
Fusion Protein as a Potential Marker for Eukaryotic Cells.
Gene 114, 239-243
Brake, AJ, Merryweather, JP, Coit, DG, Heberlein, UA, Masiarz, GR, Mullenbach, GT, Urdea, MS,
Valenzuela, P., and Barr, PJ (1984) a-Factor- Directed Synthesis and Secretion of Mature Foreign Proteins in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 81, 4642- 4646
Calmels, T., Parriche, M., Burand, H., and Tiraby, G. (1991) High Efficiency Transformation of Tolypocladium geodes Conidiospores to Phleomycin Resistance. Curr. Genet. 20, 309- 314
Cregg, JM, Barringer, KJ, and Hessler, AY (1985) Pichia pastoris as a Host System for Transformations. Mol. Cell. Biol. 5, 3376-3385
Cregg, JM, Madden, KR, Barringer, KJ, Thill, G., and Stillman, CA (1989) Functional Characterization of the
Two Alcohol Oxidase Genes from the Yeast, Pichia pastoris. Mol. Cell. Biol. 9, 1316-1323
Drocourt, D., Calmels, TPG, Reynes, JP, Baron, M., and Tiraby, G. (1990) Cassettes of the Streptoalloteichus hindustanus ble Gene for Transformation of Lower and Higher Eukaryotes to Phleomycin Resistance. Nucleic Acids Res. 18, 4009
Evan, GT, Lewis, GK, Ramsay, G., and Bishop, VM (1985) Isolation of Monoclonal Antibodies Specific for c-myc Proto-oncogene Product. Mol. Cell. Biol. 5, 3610-3616 Gatignol, A., Durand, H., and Tiraby, G. (1988) Bleomycin Resistance Conferred by a Drug-binding Protein. FEB Letters 230, 171-175
Gietz, RD, and Schiestl, RH (1996) in Methods in Molecular and Cellular Biology, in press
Henikoff, S., and Cohen, EH (1984) Sequences Responsible for Transcription Termination on a Gene Segment in Saccharomyces cerevisiae. Mol. Cell. Biol. 4, 1515-1520
Irniger, S., Egli, CM, and Braus, GH (1991) Different Classes of Polyadenylation Sites in the Yeast Saccharomyces cerevisiae. Mol. Cell. Bio. 11, 3060-3069
Kozak, M. (1987) An Analysis of 5AL-Noncoding Sequences from 699 Vertebrate Messenger RNAs . Nucleic Acids Res.
15, 8125-8148
Kozak, M. (1990) Downstream Secondary Structure Facilitates Recognition of Initiator Codons by Eukaryotic
Ribosomes. Proc. Natl. Acad. Sci. USA 87, 8301-8305
Kozak, M. (1991) An Analysis of Vertebrate mRNA Sequences: Intimations of Translational Control. J. Cell Biology 115, 887-903
Mulsant, P., Tiraby, G., Kallerhoff, J., and Perret, J. (1988) Phleomycin Resistance as a Dominant Selectable Marker in CHO Cells. Somat. Cell Mol. Genet. 14, 243-252
Perez, P., Tiraby, G., Kallerhoff, J., and Perret, J. (1989) Phleomycin Resistance as a Dominant Selectable Marker for Plant Cell Transformation. Plant Mol. Biol. 13, 365-373
Sambrook, J., Fritsch, EE, and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Second Ed., Cold Spring
Harbor Laboratory Press, Plainview, New York
Scorer, CA, Buckholz, RG , Clare, JJ, and Romanos, MA (1993) The Intracellular Production and Secretion of
HIV-1 Envelope Protein in the Methylotrophic Yeast Pichia pastoris. Gene 136,
111-119 Waterham, HR, Digan, ME, Koutz, PJ, Lair, SV, and Cregg, JM (1997) Isolation of the Pichia pastoris
Glyceraldehyde-3-Phosphate Dehydrogenase Gene and Regulation and Use of its Promoter. Gene 186, 37-44
Zaret, KS, and Sherman, F. (1984) Mutationally Altered 3AL Ends of Yeast CYC1 mRNA Affect Transcript Stability and Translational Efficiency. J. Mol. Biol. 177, 107-136
Thi Lan Than Bien et al. J. Gen. Appl . Microb. 175-182 (2014)
Claims
1. A protein encoded by the sequence having SEQ ID No: 1 and / or by a sequence having at least 95% sequence identity with SEQ ID no: 1 for use in the medical field.
2. A protein having amino acid sequence of SEQ ID No: 2 and / or amino acid sequence having at least 95% sequence identity with SEQ ID no: 2 for use in the medical field.
3. Composition comprising the protein according to one of claims 1 or 2 for use in the medical field.
4. Composition according to claim 3 further comprising at least one of saline buffer preferably PBS, protease inhibitor, MgC12, pharmaceutically acceptable excipients, pharmaceutically acceptable carriers, pharmaceutically acceptable thickeners and gelling agents, pharmaceutically acceptable carriers, preferably lipid, even more preferably liposomes, pharmaceutically acceptable adjuvants, cellulose preferably methylcellulose, a lysate of bacterial or fungal cells expressing said protein, preferably of pichia pastoris or E. coli, an antiviral drug.
5. Composition according to claim 3 or 4 formulated in spray, semi-liquid, creamy, semi-solid, solid, cream, suspension, milk, or gel form.
6. Protein or compositions according to any one of claims 1 to 5 for use in the treatment of viral infections, preferably Sars-Cov-2 .
7. Disinfectant composition comprising a protein having amino acid sequence of SEQ ID No: 2 and / or amino acid sequence having at least 95% sequence identity with SEQ ID No: 2.
8. Disinfectant composition according to claim 7 further comprising at least one of saline buffer preferably PBS, protease inhibitor, MgC12, excipients, carriers, thickeners and gelling agents, preferably methyl orixane or alginates such as calcium or sodium alginate, carriers, adjuvants,
cellulose preferably methylcellulose , a lysate of bacterial or fungal cells , preferably of pichia pastoris or E . coli .
9 . Method for the control or elimination of viruses , preferably Sars-CoV-2 , from environments or surfaces where this method includes the application on the surface or parts of it of at least one between
- a lysate of microorganisms , preferably pichia pastoris or E . coli , expressing the protein having SEQ ID no 2 and / or a protein having at least 95% sequence identity with SEQ ID no 2
- a protein having SEQ ID no 2 and / or a protein having at least the 95% sequence identity with SEQ ID No . 2
- compositions according to any one of claims 7 and 8 .
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| IT102021000023531 | 2021-09-13 | ||
| IT202100023531 | 2021-09-13 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2023037329A1 true WO2023037329A1 (en) | 2023-03-16 |
Family
ID=78649959
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/IB2022/058567 WO2023037329A1 (en) | 2021-09-13 | 2022-09-12 | Synthetic protein for use in the medical field |
Country Status (1)
| Country | Link |
|---|---|
| WO (1) | WO2023037329A1 (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021181355A1 (en) * | 2020-03-13 | 2021-09-16 | Delphinus Biotech S.R.L. | Antimicrobial protein and relative uses |
| WO2021181353A1 (en) * | 2020-03-13 | 2021-09-16 | Delphinus Biotech S.R.L. | Antimicrobial protein for use in the medical field |
-
2022
- 2022-09-12 WO PCT/IB2022/058567 patent/WO2023037329A1/en unknown
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021181355A1 (en) * | 2020-03-13 | 2021-09-16 | Delphinus Biotech S.R.L. | Antimicrobial protein and relative uses |
| WO2021181353A1 (en) * | 2020-03-13 | 2021-09-16 | Delphinus Biotech S.R.L. | Antimicrobial protein for use in the medical field |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Ibrahim et al. | Strategies for new antimicrobial proteins and peptides: lysozyme and aprotinin as model molecules | |
| US20190315642A1 (en) | Compositions for in vivo Expression of Therapeutic Sequences in the Microbiome | |
| CN110029111A (en) | Pichia pastoris produces the single-stranded method of recombination human source typeⅡ Collagen | |
| US9932563B2 (en) | Compositions and methods for inhibiting gene expressions | |
| US9676641B2 (en) | Therapeutic phages and methods for delivery of nucleic acids for therapeutic uses | |
| WO1996003519A1 (en) | Ubiquitin-lytic peptide fusion gene constructs, protein products deriving therefrom, and methods of making and using same | |
| US20230242597A1 (en) | Antimicrobial protein for use in the medical field | |
| US20230242921A1 (en) | Antimicrobial protein and relative use | |
| WO2023037329A1 (en) | Synthetic protein for use in the medical field | |
| ES2219752T3 (en) | ENZYM WITH PECTIN ESTERASE ACTIVITY. | |
| US20230242920A1 (en) | Antimicrobial protein and related use in agriculture | |
| KR102104121B1 (en) | An anti-microbial and anti-fungal peptide, Aranetoxin-Ab2a isolated from Argiope bruennichi and uses thereof | |
| IT202100002597A1 (en) | ANTIMICROBIAL PROTEIN AND ITS USES | |
| IT202100002606A1 (en) | ANTIMICROBIAL PROTEIN FOR MEDICAL USE | |
| KR102353992B1 (en) | Lactobacillus plantarum NIBR K9 Having Anti-Microbial and Anti-Viral Activity, Anti-Microbial and Anti-Viral Peptides Isolated from the Same, Uses thereof | |
| EP1091754A1 (en) | Crosslink-stabilized indolicidin analogs | |
| CN118064410A (en) | Antiviral protease CPAVM1 and application thereof | |
| KR102311729B1 (en) | Anti-Microbial Peptide Plantaricin, Isolated from Lactobacillus Plantarum and Uses thereof | |
| KR20020067108A (en) | Transducing domain, transducing domain-cargo molecule complex and uses thereof | |
| CN113943724A (en) | Human lysozyme-defensin fusion protein and construction method thereof | |
| Rathi | Purification and phytotoxic analysis of ceratoplatanin proteins in Botrytis cinerea | |
| KR20020010445A (en) | Cell-transducing HIV-1 Tat-superoxide dismutase fusion protein and use of the fusion protein | |
| KR20200031721A (en) | An anti-microbial and anti-fungal peptide, Aranetoxin-Ab3a isolated from Argiope bruennichi and uses thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| 121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 22786839 Country of ref document: EP Kind code of ref document: A1 |
|
| NENP | Non-entry into the national phase |
Ref country code: DE |