KR20230143693A - Recombinant Stx2e Protein, Mass Production Method thereof, and Swine Edema Disease Vaccine Composition comprising the same - Google Patents
Recombinant Stx2e Protein, Mass Production Method thereof, and Swine Edema Disease Vaccine Composition comprising the same Download PDFInfo
- Publication number
- KR20230143693A KR20230143693A KR1020220042625A KR20220042625A KR20230143693A KR 20230143693 A KR20230143693 A KR 20230143693A KR 1020220042625 A KR1020220042625 A KR 1020220042625A KR 20220042625 A KR20220042625 A KR 20220042625A KR 20230143693 A KR20230143693 A KR 20230143693A
- Authority
- KR
- South Korea
- Prior art keywords
- recombinant
- recombinant protein
- vaccine composition
- edema disease
- protein
- Prior art date
Links
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 title claims abstract description 47
- 206010030113 Oedema Diseases 0.000 title claims abstract description 44
- 239000000203 mixture Substances 0.000 title claims abstract description 38
- 229960005486 vaccine Drugs 0.000 title claims abstract description 34
- 241000282898 Sus scrofa Species 0.000 title claims abstract description 25
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 9
- 108090000623 proteins and genes Proteins 0.000 title abstract description 32
- 102000004169 proteins and genes Human genes 0.000 title abstract description 30
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 claims abstract description 37
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 claims abstract description 37
- 238000000034 method Methods 0.000 claims abstract description 14
- 230000036039 immunity Effects 0.000 claims abstract description 8
- 241000588724 Escherichia coli Species 0.000 claims description 15
- 239000002671 adjuvant Substances 0.000 claims description 13
- 239000013604 expression vector Substances 0.000 claims description 12
- 238000003259 recombinant expression Methods 0.000 claims description 12
- WNROFYMDJYEPJX-UHFFFAOYSA-K aluminium hydroxide Chemical compound [OH-].[OH-].[OH-].[Al+3] WNROFYMDJYEPJX-UHFFFAOYSA-K 0.000 claims description 8
- 238000000746 purification Methods 0.000 claims description 8
- 102000035195 Peptidases Human genes 0.000 claims description 6
- 108091005804 Peptidases Proteins 0.000 claims description 6
- 235000019833 protease Nutrition 0.000 claims description 6
- 239000003674 animal food additive Substances 0.000 claims description 5
- 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 4
- ILAYIAGXTHKHNT-UHFFFAOYSA-N 4-[4-(2,4,6-trimethyl-phenylamino)-pyrimidin-2-ylamino]-benzonitrile Chemical compound CC1=CC(C)=CC(C)=C1NC1=CC=NC(NC=2C=CC(=CC=2)C#N)=N1 ILAYIAGXTHKHNT-UHFFFAOYSA-N 0.000 claims description 3
- 108091034117 Oligonucleotide Proteins 0.000 claims description 3
- 229940123384 Toll-like receptor (TLR) agonist Drugs 0.000 claims description 3
- 229940037003 alum Drugs 0.000 claims description 3
- 230000002708 enhancing effect Effects 0.000 claims description 3
- 230000028993 immune response Effects 0.000 claims description 3
- 230000003308 immunostimulating effect Effects 0.000 claims description 3
- 230000001939 inductive effect Effects 0.000 claims description 3
- 239000003970 toll like receptor agonist Substances 0.000 claims description 3
- 239000007762 w/o emulsion Substances 0.000 claims description 3
- 238000012545 processing Methods 0.000 claims description 2
- 230000001131 transforming effect Effects 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims 2
- 238000001814 protein method Methods 0.000 claims 1
- 241000282887 Suidae Species 0.000 abstract description 5
- 150000001413 amino acids Chemical group 0.000 description 9
- 208000024891 symptom Diseases 0.000 description 8
- 238000002360 preparation method Methods 0.000 description 7
- 108020004414 DNA Proteins 0.000 description 6
- 239000003242 anti bacterial agent Substances 0.000 description 6
- 201000010099 disease Diseases 0.000 description 6
- 238000009472 formulation Methods 0.000 description 6
- 239000013612 plasmid Substances 0.000 description 6
- 230000000694 effects Effects 0.000 description 5
- 208000015181 infectious disease Diseases 0.000 description 5
- 239000000546 pharmaceutical excipient Substances 0.000 description 5
- 239000000725 suspension Substances 0.000 description 5
- 108700012359 toxins Proteins 0.000 description 5
- 238000011282 treatment Methods 0.000 description 5
- 229940088710 antibiotic agent Drugs 0.000 description 4
- 239000003085 diluting agent Substances 0.000 description 4
- 210000002966 serum Anatomy 0.000 description 4
- 239000003381 stabilizer Substances 0.000 description 4
- 239000003053 toxin Substances 0.000 description 4
- 231100000765 toxin Toxicity 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 3
- 238000002965 ELISA Methods 0.000 description 3
- 108091028043 Nucleic acid sequence Proteins 0.000 description 3
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 description 3
- 229920002472 Starch Polymers 0.000 description 3
- 108091007433 antigens Proteins 0.000 description 3
- 102000036639 antigens Human genes 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 238000012790 confirmation Methods 0.000 description 3
- 230000034994 death Effects 0.000 description 3
- 239000000839 emulsion Substances 0.000 description 3
- 239000008187 granular material Substances 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 150000007523 nucleic acids Chemical group 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 239000003755 preservative agent Substances 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 3
- 239000008107 starch Substances 0.000 description 3
- 235000019698 starch Nutrition 0.000 description 3
- 229940032147 starch Drugs 0.000 description 3
- 238000002255 vaccination Methods 0.000 description 3
- 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 2
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- 208000035143 Bacterial infection Diseases 0.000 description 2
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 2
- 206010012735 Diarrhoea Diseases 0.000 description 2
- 239000004606 Fillers/Extenders Substances 0.000 description 2
- 208000007212 Foot-and-Mouth Disease Diseases 0.000 description 2
- 241000710198 Foot-and-mouth disease virus Species 0.000 description 2
- 108010010803 Gelatin Proteins 0.000 description 2
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-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
- 239000006137 Luria-Bertani broth Substances 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 229910019142 PO4 Inorganic materials 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
- 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 2
- WNLRTRBMVRJNCN-UHFFFAOYSA-N adipic acid Chemical compound OC(=O)CCCCC(O)=O WNLRTRBMVRJNCN-UHFFFAOYSA-N 0.000 description 2
- 229960000723 ampicillin Drugs 0.000 description 2
- 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 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 208000022362 bacterial infectious disease Diseases 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 239000002775 capsule Substances 0.000 description 2
- 239000000969 carrier Substances 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 235000005911 diet Nutrition 0.000 description 2
- 230000037213 diet Effects 0.000 description 2
- 208000010643 digestive system disease Diseases 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000003995 emulsifying agent Substances 0.000 description 2
- 238000009313 farming Methods 0.000 description 2
- 210000001035 gastrointestinal tract Anatomy 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 239000008273 gelatin Substances 0.000 description 2
- 229920000159 gelatin Polymers 0.000 description 2
- 235000019322 gelatine Nutrition 0.000 description 2
- 235000011852 gelatine desserts Nutrition 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 230000017730 intein-mediated protein splicing Effects 0.000 description 2
- 238000005342 ion exchange 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
- 229930027917 kanamycin Natural products 0.000 description 2
- 229960000318 kanamycin Drugs 0.000 description 2
- 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 2
- 229930182823 kanamycin A Natural products 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 239000008101 lactose Substances 0.000 description 2
- 229960001375 lactose Drugs 0.000 description 2
- 244000144972 livestock Species 0.000 description 2
- HQKMJHAJHXVSDF-UHFFFAOYSA-L magnesium stearate Chemical compound [Mg+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O HQKMJHAJHXVSDF-UHFFFAOYSA-L 0.000 description 2
- -1 pH adjusters Substances 0.000 description 2
- 230000001717 pathogenic effect Effects 0.000 description 2
- 235000021317 phosphate Nutrition 0.000 description 2
- 230000003449 preventive effect Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000002441 reversible effect Effects 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 229960004793 sucrose Drugs 0.000 description 2
- 239000004094 surface-active agent Substances 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
- 239000000454 talc Substances 0.000 description 2
- 229910052623 talc Inorganic materials 0.000 description 2
- 235000012222 talc Nutrition 0.000 description 2
- 108010061238 threonyl-glycine Proteins 0.000 description 2
- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 230000004584 weight gain Effects 0.000 description 2
- 235000019786 weight gain Nutrition 0.000 description 2
- 239000000080 wetting agent Substances 0.000 description 2
- GVJHHUAWPYXKBD-IEOSBIPESA-N α-tocopherol Chemical compound OC1=C(C)C(C)=C2O[C@@](CCC[C@H](C)CCC[C@H](C)CCCC(C)C)(C)CCC2=C1C GVJHHUAWPYXKBD-IEOSBIPESA-N 0.000 description 2
- PFTAWBLQPZVEMU-DZGCQCFKSA-N (+)-catechin Chemical compound C1([C@H]2OC3=CC(O)=CC(O)=C3C[C@@H]2O)=CC=C(O)C(O)=C1 PFTAWBLQPZVEMU-DZGCQCFKSA-N 0.000 description 1
- BJEPYKJPYRNKOW-REOHCLBHSA-N (S)-malic acid Chemical compound OC(=O)[C@@H](O)CC(O)=O BJEPYKJPYRNKOW-REOHCLBHSA-N 0.000 description 1
- GEYOCULIXLDCMW-UHFFFAOYSA-N 1,2-phenylenediamine Chemical compound NC1=CC=CC=C1N GEYOCULIXLDCMW-UHFFFAOYSA-N 0.000 description 1
- TUSDEZXZIZRFGC-UHFFFAOYSA-N 1-O-galloyl-3,6-(R)-HHDP-beta-D-glucose Natural products OC1C(O2)COC(=O)C3=CC(O)=C(O)C(O)=C3C3=C(O)C(O)=C(O)C=C3C(=O)OC1C(O)C2OC(=O)C1=CC(O)=C(O)C(O)=C1 TUSDEZXZIZRFGC-UHFFFAOYSA-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
- NIXOWILDQLNWCW-UHFFFAOYSA-N Acrylic acid Chemical group OC(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 description 1
- PIPTUBPKYFRLCP-NHCYSSNCSA-N Ala-Ala-Phe Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 PIPTUBPKYFRLCP-NHCYSSNCSA-N 0.000 description 1
- YIGLXQRFQVWFEY-NRPADANISA-N Ala-Gln-Val Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C(C)C)C(O)=O YIGLXQRFQVWFEY-NRPADANISA-N 0.000 description 1
- OMFMCIVBKCEMAK-CYDGBPFRSA-N Ala-Leu-Val-Ser Chemical compound C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(O)=O OMFMCIVBKCEMAK-CYDGBPFRSA-N 0.000 description 1
- XSLGWYYNOSUMRM-ZKWXMUAHSA-N Ala-Val-Asn Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O XSLGWYYNOSUMRM-ZKWXMUAHSA-N 0.000 description 1
- LYILPUNCKACNGF-NAKRPEOUSA-N Ala-Val-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](C)N LYILPUNCKACNGF-NAKRPEOUSA-N 0.000 description 1
- AUFHLLPVPSMEOG-YUMQZZPRSA-N Arg-Gly-Glu Chemical compound NC(N)=NCCC[C@H](N)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(O)=O AUFHLLPVPSMEOG-YUMQZZPRSA-N 0.000 description 1
- GXXWTNKNFFKTJB-NAKRPEOUSA-N Arg-Ile-Ser Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(O)=O GXXWTNKNFFKTJB-NAKRPEOUSA-N 0.000 description 1
- CQMQJWRCRQSBAF-BPUTZDHNSA-N Asn-Arg-Trp Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(=O)N)N CQMQJWRCRQSBAF-BPUTZDHNSA-N 0.000 description 1
- IOTKDTZEEBZNCM-UGYAYLCHSA-N Asn-Asn-Ile Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O IOTKDTZEEBZNCM-UGYAYLCHSA-N 0.000 description 1
- HFPXZWPUVFVNLL-GUBZILKMSA-N Asn-Leu-Gln Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O HFPXZWPUVFVNLL-GUBZILKMSA-N 0.000 description 1
- DAYDURRBMDCCFL-AAEUAGOBSA-N Asn-Trp-Gly Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)NCC(=O)O)NC(=O)[C@H](CC(=O)N)N DAYDURRBMDCCFL-AAEUAGOBSA-N 0.000 description 1
- CBHVAFXKOYAHOY-NHCYSSNCSA-N Asn-Val-Leu Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O CBHVAFXKOYAHOY-NHCYSSNCSA-N 0.000 description 1
- DBWYWXNMZZYIRY-LPEHRKFASA-N Asp-Arg-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(=O)O)N)C(=O)O DBWYWXNMZZYIRY-LPEHRKFASA-N 0.000 description 1
- AMRANMVXQWXNAH-ZLUOBGJFSA-N Asp-Cys-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](CS)NC(=O)[C@@H](N)CC(O)=O AMRANMVXQWXNAH-ZLUOBGJFSA-N 0.000 description 1
- ORRJQLIATJDMQM-HJGDQZAQSA-N Asp-Leu-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CC(O)=O ORRJQLIATJDMQM-HJGDQZAQSA-N 0.000 description 1
- 238000009010 Bradford assay Methods 0.000 description 1
- YDNKGFDKKRUKPY-JHOUSYSJSA-N C16 ceramide Natural products CCCCCCCCCCCCCCCC(=O)N[C@@H](CO)[C@H](O)C=CCCCCCCCCCCCCC YDNKGFDKKRUKPY-JHOUSYSJSA-N 0.000 description 1
- PTHCMJGKKRQCBF-UHFFFAOYSA-N Cellulose, microcrystalline Chemical compound OC1C(O)C(OC)OC(CO)C1OC1C(O)C(O)C(OC)C(CO)O1 PTHCMJGKKRQCBF-UHFFFAOYSA-N 0.000 description 1
- 229920001661 Chitosan Polymers 0.000 description 1
- 102000009016 Cholera Toxin Human genes 0.000 description 1
- 108010049048 Cholera Toxin Proteins 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- RFHGRMMADHHQSA-KBIXCLLPSA-N Cys-Gln-Ile Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O RFHGRMMADHHQSA-KBIXCLLPSA-N 0.000 description 1
- YNJBLTDKTMKEET-ZLUOBGJFSA-N Cys-Ser-Ser Chemical compound SC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O YNJBLTDKTMKEET-ZLUOBGJFSA-N 0.000 description 1
- 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 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
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-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
- LVGKNOAMLMIIKO-UHFFFAOYSA-N Elaidinsaeure-aethylester Natural products CCCCCCCCC=CCCCCCCCC(=O)OCC LVGKNOAMLMIIKO-UHFFFAOYSA-N 0.000 description 1
- 241000305071 Enterobacterales Species 0.000 description 1
- 206010015993 Eyelid oedema Diseases 0.000 description 1
- 239000001263 FEMA 3042 Substances 0.000 description 1
- MLSKFHLRFVGNLL-WDCWCFNPSA-N Gln-Leu-Thr Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O MLSKFHLRFVGNLL-WDCWCFNPSA-N 0.000 description 1
- HMIXCETWRYDVMO-GUBZILKMSA-N Gln-Pro-Glu Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(O)=O HMIXCETWRYDVMO-GUBZILKMSA-N 0.000 description 1
- KUBFPYIMAGXGBT-ACZMJKKPSA-N Gln-Ser-Ala Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(O)=O KUBFPYIMAGXGBT-ACZMJKKPSA-N 0.000 description 1
- ZOXBSICWUDAOHX-GUBZILKMSA-N Glu-Asn-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](N)CCC(O)=O ZOXBSICWUDAOHX-GUBZILKMSA-N 0.000 description 1
- NTBDVNJIWCKURJ-ACZMJKKPSA-N Glu-Asp-Asn Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O NTBDVNJIWCKURJ-ACZMJKKPSA-N 0.000 description 1
- WATXSTJXNBOHKD-LAEOZQHASA-N Glu-Asp-Val Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O WATXSTJXNBOHKD-LAEOZQHASA-N 0.000 description 1
- IQACOVZVOMVILH-FXQIFTODSA-N Glu-Glu-Ser Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(O)=O IQACOVZVOMVILH-FXQIFTODSA-N 0.000 description 1
- ALMBZBOCGSVSAI-ACZMJKKPSA-N Glu-Ser-Asn Chemical compound C(CC(=O)O)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(=O)N)C(=O)O)N ALMBZBOCGSVSAI-ACZMJKKPSA-N 0.000 description 1
- QLNKFGTZOBVMCS-JBACZVJFSA-N Glu-Tyr-Trp Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O QLNKFGTZOBVMCS-JBACZVJFSA-N 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
- PUUYVMYCMIWHFE-BQBZGAKWSA-N Gly-Ala-Arg Chemical compound NCC(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N PUUYVMYCMIWHFE-BQBZGAKWSA-N 0.000 description 1
- SOEGEPHNZOISMT-BYPYZUCNSA-N Gly-Ser-Gly Chemical compound NCC(=O)N[C@@H](CO)C(=O)NCC(O)=O SOEGEPHNZOISMT-BYPYZUCNSA-N 0.000 description 1
- CUVBTVWFVIIDOC-YEPSODPASA-N Gly-Thr-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)CN CUVBTVWFVIIDOC-YEPSODPASA-N 0.000 description 1
- GJHWILMUOANXTG-WPRPVWTQSA-N Gly-Val-Arg Chemical compound [H]NCC(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O GJHWILMUOANXTG-WPRPVWTQSA-N 0.000 description 1
- 102000002068 Glycopeptides Human genes 0.000 description 1
- 108010015899 Glycopeptides Proteins 0.000 description 1
- 229920000084 Gum arabic Polymers 0.000 description 1
- JBCLFWXMTIKCCB-UHFFFAOYSA-N H-Gly-Phe-OH Natural products NCC(=O)NC(C(O)=O)CC1=CC=CC=C1 JBCLFWXMTIKCCB-UHFFFAOYSA-N 0.000 description 1
- IDQNVIWPPWAFSY-AVGNSLFASA-N His-His-Gln Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CCC(N)=O)C(O)=O IDQNVIWPPWAFSY-AVGNSLFASA-N 0.000 description 1
- QYZYJFXHXYUZMZ-UGYAYLCHSA-N Ile-Asn-Asn Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CC(=O)N)C(=O)O)N QYZYJFXHXYUZMZ-UGYAYLCHSA-N 0.000 description 1
- FUOYNOXRWPJPAN-QEWYBTABSA-N Ile-Glu-Phe Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)N FUOYNOXRWPJPAN-QEWYBTABSA-N 0.000 description 1
- ZDNNDIJTUHQCAM-MXAVVETBSA-N Ile-Ser-Phe Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)N ZDNNDIJTUHQCAM-MXAVVETBSA-N 0.000 description 1
- IMQLKJBTEOYOSI-GPIVLXJGSA-N Inositol-hexakisphosphate Chemical compound OP(O)(=O)O[C@H]1[C@H](OP(O)(O)=O)[C@@H](OP(O)(O)=O)[C@H](OP(O)(O)=O)[C@H](OP(O)(O)=O)[C@@H]1OP(O)(O)=O IMQLKJBTEOYOSI-GPIVLXJGSA-N 0.000 description 1
- LHSGPCFBGJHPCY-UHFFFAOYSA-N L-leucine-L-tyrosine Natural products CC(C)CC(N)C(=O)NC(C(O)=O)CC1=CC=C(O)C=C1 LHSGPCFBGJHPCY-UHFFFAOYSA-N 0.000 description 1
- 241000880493 Leptailurus serval Species 0.000 description 1
- IGUOAYLTQJLPPD-DCAQKATOSA-N Leu-Asn-Arg Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(O)=O)CCCN=C(N)N IGUOAYLTQJLPPD-DCAQKATOSA-N 0.000 description 1
- BAJIJEGGUYXZGC-CIUDSAMLSA-N Leu-Asn-Cys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CS)C(=O)O)N BAJIJEGGUYXZGC-CIUDSAMLSA-N 0.000 description 1
- CQGSYZCULZMEDE-UHFFFAOYSA-N Leu-Gln-Pro Natural products CC(C)CC(N)C(=O)NC(CCC(N)=O)C(=O)N1CCCC1C(O)=O CQGSYZCULZMEDE-UHFFFAOYSA-N 0.000 description 1
- 239000006142 Luria-Bertani Agar Substances 0.000 description 1
- NKKFVJRLCCUJNA-QWRGUYRKSA-N Lys-Gly-Lys Chemical compound NCCCC[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCCCN NKKFVJRLCCUJNA-QWRGUYRKSA-N 0.000 description 1
- PGIAYBDRSREMJY-UHFFFAOYSA-N Lys-Lys-Met-Phe Chemical compound NCCCCC(N)C(=O)NC(CCCCN)C(=O)NC(CCSC)C(=O)NC(C(O)=O)CC1=CC=CC=C1 PGIAYBDRSREMJY-UHFFFAOYSA-N 0.000 description 1
- ODTZHNZPINULEU-KKUMJFAQSA-N Lys-Phe-Asn Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](CCCCN)N ODTZHNZPINULEU-KKUMJFAQSA-N 0.000 description 1
- DNWBUCHHMRQWCZ-GUBZILKMSA-N Lys-Ser-Gln Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCC(N)=O DNWBUCHHMRQWCZ-GUBZILKMSA-N 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
- YRAWWKUTNBILNT-FXQIFTODSA-N Met-Ala-Ala Chemical compound CSCC[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(O)=O YRAWWKUTNBILNT-FXQIFTODSA-N 0.000 description 1
- NDJSSFWDYDUQID-YTWAJWBKSA-N Met-Thr-Pro Chemical compound C[C@H]([C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CCSC)N)O NDJSSFWDYDUQID-YTWAJWBKSA-N 0.000 description 1
- ZBLSZPYQQRIHQU-RCWTZXSCSA-N Met-Thr-Val Chemical compound CSCC[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(O)=O ZBLSZPYQQRIHQU-RCWTZXSCSA-N 0.000 description 1
- 229920000168 Microcrystalline cellulose Polymers 0.000 description 1
- CRJGESKKUOMBCT-VQTJNVASSA-N N-acetylsphinganine Chemical compound CCCCCCCCCCCCCCC[C@@H](O)[C@H](CO)NC(C)=O CRJGESKKUOMBCT-VQTJNVASSA-N 0.000 description 1
- 206010060860 Neurological symptom Diseases 0.000 description 1
- 101100342977 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) leu-1 gene Proteins 0.000 description 1
- LRBQNJMCXXYXIU-PPKXGCFTSA-N Penta-digallate-beta-D-glucose Natural products OC1=C(O)C(O)=CC(C(=O)OC=2C(=C(O)C=C(C=2)C(=O)OC[C@@H]2[C@H]([C@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)O2)OC(=O)C=2C=C(OC(=O)C=3C=C(O)C(O)=C(O)C=3)C(O)=C(O)C=2)O)=C1 LRBQNJMCXXYXIU-PPKXGCFTSA-N 0.000 description 1
- IMQLKJBTEOYOSI-UHFFFAOYSA-N Phytic acid Natural products OP(O)(=O)OC1C(OP(O)(O)=O)C(OP(O)(O)=O)C(OP(O)(O)=O)C(OP(O)(O)=O)C1OP(O)(O)=O IMQLKJBTEOYOSI-UHFFFAOYSA-N 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- QGOZJLYCGRYYRW-KKUMJFAQSA-N Pro-Glu-Tyr Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O QGOZJLYCGRYYRW-KKUMJFAQSA-N 0.000 description 1
- XYSXOCIWCPFOCG-IHRRRGAJSA-N Pro-Leu-Leu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O XYSXOCIWCPFOCG-IHRRRGAJSA-N 0.000 description 1
- 102000002278 Ribosomal Proteins Human genes 0.000 description 1
- 108010000605 Ribosomal Proteins Proteins 0.000 description 1
- 101710158076 Ribosome-inactivating protein Proteins 0.000 description 1
- WTWGOQRNRFHFQD-JBDRJPRFSA-N Ser-Ala-Ile Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O WTWGOQRNRFHFQD-JBDRJPRFSA-N 0.000 description 1
- UGJRQLURDVGULT-LKXGYXEUSA-N Ser-Asn-Thr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O UGJRQLURDVGULT-LKXGYXEUSA-N 0.000 description 1
- AEGUWTFAQQWVLC-BQBZGAKWSA-N Ser-Gly-Arg Chemical compound [H]N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(O)=O AEGUWTFAQQWVLC-BQBZGAKWSA-N 0.000 description 1
- JWOBLHJRDADHLN-KKUMJFAQSA-N Ser-Leu-Tyr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O JWOBLHJRDADHLN-KKUMJFAQSA-N 0.000 description 1
- QJKPECIAWNNKIT-KKUMJFAQSA-N Ser-Lys-Tyr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O QJKPECIAWNNKIT-KKUMJFAQSA-N 0.000 description 1
- GMXIJHCBTZDAPD-QPHKQPEJSA-N Thr-Ile-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)O)NC(=O)[C@H]([C@@H](C)O)N GMXIJHCBTZDAPD-QPHKQPEJSA-N 0.000 description 1
- ISLDRLHVPXABBC-IEGACIPQSA-N Thr-Leu-Trp Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O ISLDRLHVPXABBC-IEGACIPQSA-N 0.000 description 1
- MXNAOGFNFNKUPD-JHYOHUSXSA-N Thr-Phe-Thr Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)O)C(O)=O MXNAOGFNFNKUPD-JHYOHUSXSA-N 0.000 description 1
- UQCNIMDPYICBTR-KYNKHSRBSA-N Thr-Thr-Gly Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(O)=O UQCNIMDPYICBTR-KYNKHSRBSA-N 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- FDKDGFGTHGJKNV-FHWLQOOXSA-N Tyr-Phe-Gln Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)O)NC(=O)[C@H](CC2=CC=C(C=C2)O)N FDKDGFGTHGJKNV-FHWLQOOXSA-N 0.000 description 1
- QPZMOUMNTGTEFR-ZKWXMUAHSA-N Val-Asn-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](C(C)C)N QPZMOUMNTGTEFR-ZKWXMUAHSA-N 0.000 description 1
- JTWIMNMUYLQNPI-WPRPVWTQSA-N Val-Gly-Arg Chemical compound CC(C)[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCCNC(N)=N JTWIMNMUYLQNPI-WPRPVWTQSA-N 0.000 description 1
- SDUBQHUJJWQTEU-XUXIUFHCSA-N Val-Ile-Lys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](C(C)C)N SDUBQHUJJWQTEU-XUXIUFHCSA-N 0.000 description 1
- ZZGPVSZDZQRJQY-ULQDDVLXSA-N Val-Leu-Phe Chemical compound CC(C)C[C@H](NC(=O)[C@@H](N)C(C)C)C(=O)N[C@@H](Cc1ccccc1)C(O)=O ZZGPVSZDZQRJQY-ULQDDVLXSA-N 0.000 description 1
- VPGCVZRRBYOGCD-AVGNSLFASA-N Val-Lys-Val Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(O)=O VPGCVZRRBYOGCD-AVGNSLFASA-N 0.000 description 1
- 229930003268 Vitamin C Natural products 0.000 description 1
- TVXBFESIOXBWNM-UHFFFAOYSA-N Xylitol Natural products OCCC(O)C(O)C(O)CCO TVXBFESIOXBWNM-UHFFFAOYSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 235000010489 acacia gum Nutrition 0.000 description 1
- 239000000205 acacia gum Substances 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 239000001361 adipic acid Substances 0.000 description 1
- 235000011037 adipic acid Nutrition 0.000 description 1
- 239000000443 aerosol Substances 0.000 description 1
- 239000000556 agonist Substances 0.000 description 1
- 108010044940 alanylglutamine Proteins 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
- 229940087168 alpha tocopherol Drugs 0.000 description 1
- BJEPYKJPYRNKOW-UHFFFAOYSA-N alpha-hydroxysuccinic acid Natural products OC(=O)C(O)CC(O)=O BJEPYKJPYRNKOW-UHFFFAOYSA-N 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
- ILRRQNADMUWWFW-UHFFFAOYSA-K aluminium phosphate Chemical compound O1[Al]2OP1(=O)O2 ILRRQNADMUWWFW-UHFFFAOYSA-K 0.000 description 1
- 229940024545 aluminum hydroxide Drugs 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000000845 anti-microbial effect Effects 0.000 description 1
- 239000003429 antifungal agent Substances 0.000 description 1
- 229940121375 antifungal agent Drugs 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 239000004599 antimicrobial Substances 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 108010013835 arginine glutamate Proteins 0.000 description 1
- 108010009111 arginyl-glycyl-glutamic acid Proteins 0.000 description 1
- 108010062796 arginyllysine Proteins 0.000 description 1
- 239000012752 auxiliary agent Substances 0.000 description 1
- WXNRAKRZUCLRBP-UHFFFAOYSA-N avridine Chemical compound CCCCCCCCCCCCCCCCCCN(CCCN(CCO)CCO)CCCCCCCCCCCCCCCCCC WXNRAKRZUCLRBP-UHFFFAOYSA-N 0.000 description 1
- 244000052616 bacterial pathogen Species 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
- 239000011230 binding agent Substances 0.000 description 1
- 239000003124 biologic agent Substances 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 229910000019 calcium carbonate Inorganic materials 0.000 description 1
- 229960003563 calcium carbonate Drugs 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 239000000378 calcium silicate Substances 0.000 description 1
- 229910052918 calcium silicate Inorganic materials 0.000 description 1
- 235000012241 calcium silicate Nutrition 0.000 description 1
- OYACROKNLOSFPA-UHFFFAOYSA-N calcium;dioxido(oxo)silane Chemical compound [Ca+2].[O-][Si]([O-])=O OYACROKNLOSFPA-UHFFFAOYSA-N 0.000 description 1
- 229940096529 carboxypolymethylene Drugs 0.000 description 1
- 235000005487 catechin Nutrition 0.000 description 1
- ADRVNXBAWSRFAJ-UHFFFAOYSA-N catechin Natural products OC1Cc2cc(O)cc(O)c2OC1c3ccc(O)c(O)c3 ADRVNXBAWSRFAJ-UHFFFAOYSA-N 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 235000010980 cellulose Nutrition 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 229940106189 ceramide Drugs 0.000 description 1
- ZVEQCJWYRWKARO-UHFFFAOYSA-N ceramide Natural products CCCCCCCCCCCCCCC(O)C(=O)NC(CO)C(O)C=CCCC=C(C)CCCCCCCCC ZVEQCJWYRWKARO-UHFFFAOYSA-N 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 229940045110 chitosan Drugs 0.000 description 1
- 229950001002 cianidanol Drugs 0.000 description 1
- 230000001886 ciliary effect Effects 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 239000008121 dextrose Substances 0.000 description 1
- 230000001079 digestive effect Effects 0.000 description 1
- PSLWZOIUBRXAQW-UHFFFAOYSA-M dimethyl(dioctadecyl)azanium;bromide Chemical compound [Br-].CCCCCCCCCCCCCCCCCC[N+](C)(C)CCCCCCCCCCCCCCCCCC PSLWZOIUBRXAQW-UHFFFAOYSA-M 0.000 description 1
- CQAIPTBBCVQRMD-UHFFFAOYSA-L dipotassium;phosphono phosphate Chemical compound [K+].[K+].OP(O)(=O)OP([O-])([O-])=O CQAIPTBBCVQRMD-UHFFFAOYSA-L 0.000 description 1
- 230000006806 disease prevention Effects 0.000 description 1
- 239000007884 disintegrant Substances 0.000 description 1
- 239000002612 dispersion medium Substances 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 210000001163 endosome Anatomy 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- LVGKNOAMLMIIKO-QXMHVHEDSA-N ethyl oleate Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OCC LVGKNOAMLMIIKO-QXMHVHEDSA-N 0.000 description 1
- 229940093471 ethyl oleate Drugs 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000029142 excretion Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 235000003599 food sweetener Nutrition 0.000 description 1
- 239000003205 fragrance Substances 0.000 description 1
- 229960001031 glucose Drugs 0.000 description 1
- 108010078144 glutaminyl-glycine Proteins 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- 229960005150 glycerol Drugs 0.000 description 1
- 108010081551 glycylphenylalanine Proteins 0.000 description 1
- 235000020688 green tea extract Nutrition 0.000 description 1
- 229940094952 green tea extract Drugs 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 239000007952 growth promoter Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 239000007951 isotonicity adjuster Substances 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 108010057821 leucylproline Proteins 0.000 description 1
- 108010012058 leucyltyrosine Proteins 0.000 description 1
- 229940069445 licorice extract Drugs 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 229940057995 liquid paraffin Drugs 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- 239000012139 lysis buffer Substances 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- 235000019359 magnesium stearate Nutrition 0.000 description 1
- 239000001630 malic acid Substances 0.000 description 1
- 235000011090 malic acid Nutrition 0.000 description 1
- 239000000845 maltitol Substances 0.000 description 1
- 235000010449 maltitol Nutrition 0.000 description 1
- VQHSOMBJVWLPSR-WUJBLJFYSA-N maltitol Chemical compound OC[C@H](O)[C@@H](O)[C@@H]([C@H](O)CO)O[C@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O VQHSOMBJVWLPSR-WUJBLJFYSA-N 0.000 description 1
- 229940035436 maltitol Drugs 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 229960001855 mannitol Drugs 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- HEBKCHPVOIAQTA-UHFFFAOYSA-N meso ribitol Natural products OCC(O)C(O)C(O)CO HEBKCHPVOIAQTA-UHFFFAOYSA-N 0.000 description 1
- 229920000609 methyl cellulose Polymers 0.000 description 1
- 239000001923 methylcellulose Substances 0.000 description 1
- 235000010981 methylcellulose Nutrition 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
- 235000019813 microcrystalline cellulose Nutrition 0.000 description 1
- 239000008108 microcrystalline cellulose Substances 0.000 description 1
- 229940016286 microcrystalline cellulose Drugs 0.000 description 1
- 235000010446 mineral oil Nutrition 0.000 description 1
- 239000002480 mineral oil Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 229940035032 monophosphoryl lipid a Drugs 0.000 description 1
- 210000004877 mucosa Anatomy 0.000 description 1
- 210000003097 mucus Anatomy 0.000 description 1
- VVGIYYKRAMHVLU-UHFFFAOYSA-N newbouldiamide Natural products CCCCCCCCCCCCCCCCCCCC(O)C(O)C(O)C(CO)NC(=O)CCCCCCCCCCCCCCCCC VVGIYYKRAMHVLU-UHFFFAOYSA-N 0.000 description 1
- 235000008390 olive oil Nutrition 0.000 description 1
- 239000004006 olive oil Substances 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 238000007911 parenteral administration Methods 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 108010051242 phenylalanylserine Proteins 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 1
- 239000000467 phytic acid Substances 0.000 description 1
- 229940068041 phytic acid Drugs 0.000 description 1
- 235000002949 phytic acid Nutrition 0.000 description 1
- 239000006187 pill Substances 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 150000008442 polyphenolic compounds Chemical class 0.000 description 1
- 235000013824 polyphenols Nutrition 0.000 description 1
- 239000001205 polyphosphate Substances 0.000 description 1
- 235000011176 polyphosphates Nutrition 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
- 235000015277 pork Nutrition 0.000 description 1
- OQZCJRJRGMMSGK-UHFFFAOYSA-M potassium metaphosphate Polymers [K+].[O-]P(=O)=O OQZCJRJRGMMSGK-UHFFFAOYSA-M 0.000 description 1
- 229910000160 potassium phosphate Inorganic materials 0.000 description 1
- 235000011009 potassium phosphates Nutrition 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 108010070643 prolylglutamic acid Proteins 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
- 238000001243 protein synthesis Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 229940092258 rosemary extract Drugs 0.000 description 1
- 235000020748 rosemary extract Nutrition 0.000 description 1
- 239000001233 rosmarinus officinalis l. extract Substances 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000001488 sodium phosphate Substances 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 229960002920 sorbitol Drugs 0.000 description 1
- 235000010356 sorbitol Nutrition 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000003765 sweetening agent Substances 0.000 description 1
- 229940033123 tannic acid Drugs 0.000 description 1
- 235000015523 tannic acid Nutrition 0.000 description 1
- LRBQNJMCXXYXIU-NRMVVENXSA-N tannic acid Chemical compound OC1=C(O)C(O)=CC(C(=O)OC=2C(=C(O)C=C(C=2)C(=O)OC[C@@H]2[C@H]([C@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)O2)OC(=O)C=2C=C(OC(=O)C=3C=C(O)C(O)=C(O)C=3)C(O)=C(O)C=2)O)=C1 LRBQNJMCXXYXIU-NRMVVENXSA-N 0.000 description 1
- 229920002258 tannic acid Polymers 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 229960000984 tocofersolan Drugs 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 230000014616 translation Effects 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
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 1
- 108010073969 valyllysine Proteins 0.000 description 1
- 210000003556 vascular endothelial cell Anatomy 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
- 235000015112 vegetable and seed oil Nutrition 0.000 description 1
- 239000008158 vegetable oil Substances 0.000 description 1
- 235000019154 vitamin C Nutrition 0.000 description 1
- 239000011718 vitamin C Substances 0.000 description 1
- 239000000811 xylitol Substances 0.000 description 1
- 235000010447 xylitol Nutrition 0.000 description 1
- 229960002675 xylitol Drugs 0.000 description 1
- HEBKCHPVOIAQTA-SCDXWVJYSA-N xylitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)CO HEBKCHPVOIAQTA-SCDXWVJYSA-N 0.000 description 1
- 239000002076 α-tocopherol Substances 0.000 description 1
- 235000004835 α-tocopherol Nutrition 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
- C07K14/245—Escherichia (G)
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/142—Amino acids; Derivatives thereof
- A23K20/147—Polymeric derivatives, e.g. peptides or proteins
-
- 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
- A61K39/02—Bacterial antigens
- A61K39/025—Enterobacteriales, e.g. Enterobacter
- A61K39/0258—Escherichia
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- 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
- A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
- A61K2039/552—Veterinary vaccine
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
- C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/50—Fusion polypeptide containing protease site
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2510/00—Genetically modified cells
- C12N2510/02—Cells for production
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2511/00—Cells for large scale production
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- General Health & Medical Sciences (AREA)
- Polymers & Plastics (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biomedical Technology (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Food Science & Technology (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Pharmacology & Pharmacy (AREA)
- Mycology (AREA)
- Immunology (AREA)
- Plant Pathology (AREA)
- Animal Husbandry (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
본 발명은 재조합 Stx2e 단백질, 이의 대량생산 방법 및 이를 포함하는 돼지 부종병 백신 조성물에 관한 것으로, 높은 순도로 재조합 단백질의 대량생산이 가능하며, 재조합 단백질은 현저한 항체 역가를 유도하여 돼지에게 부종병에 대한 면역능을 부여할 수 있으므로, 돼지, 특히 자돈의 부종병 예방을 위한 백신 조성물로써 유용하게 활용될 수 있다.The present invention relates to a recombinant Stx2e protein, a method for mass production thereof, and a porcine edema disease vaccine composition comprising the same. It enables mass production of the recombinant protein with high purity, and the recombinant protein induces significant antibody titers to prevent edema disease in pigs. Since it can confer immunity against swine, it can be usefully used as a vaccine composition to prevent edema disease in pigs, especially piglets.
Description
본 발명은 재조합 Stx2e 단백질, 이의 대량생산 방법 및 이를 포함하는 돼지 부종병 백신 조성물에 관한 것이다.The present invention relates to a recombinant Stx2e protein, a method for mass production thereof, and a porcine edema disease vaccine composition comprising the same.
양돈 산업에 있어 경제적 손실의 가장 큰 원인 중 하나는 자돈 시기에 소화기 질병에 의한 설사로 인한 폐사 및 사료 효율의 저하에 따른 증체율 저하의 결과이다. 이처럼 양돈 산업에 심각한 경제적 손실을 초래하는 세균성 소화기 질환을 예방 및 치료하기 위한 예방백신 및 질병 저감제를 개발하기 위해 많은 연구가 진행 중에 있다. 소화기 점막은 대다수의 질병 유발 병원성 세균과 처음으로 접하는 장소임과 동시에 이들 세균의 감염을 방어하는 최전방으로서 매우 중요한 곳이기도 하다. 최근 돼지 부종병은 계속 증가하고 있는 추세이며 이를 예방하기 위한 예방 백신 개발이 활발히 수행되고 있지만 아직 상용화되어 판매되고 있는 제품은 극히 드문 실정이다.One of the biggest causes of economic loss in the pig farming industry is the result of death due to diarrhea caused by digestive diseases during the piglet stage and decreased weight gain due to reduced feed efficiency. As such, much research is underway to develop preventive vaccines and disease reduction agents to prevent and treat bacterial digestive diseases that cause serious economic losses in the pig farming industry. The digestive mucosa is the place where we first encounter most disease-causing pathogenic bacteria, and at the same time, it is a very important place as the front line to defend against infection by these bacteria. Recently, swine edema disease has been on the rise, and preventive vaccines are being actively developed to prevent it, but products that have been commercialized and sold are extremely rare.
돼지 부종병 주로 Stx2e+ 대장균(E.coli)에 의해 주로 발병되는 것으로 보고되고 있다. 특히 돼지 부종병은 최근 구제역(Foot and Mouth Disease: FMD)와 돼지 유행성 설사병(Porcine epidemic diarrhea: PED)에 이어 양돈가에 경제적으로 막대한 경제적 소실을 주고있는 주요 세균성 질병으로 돼지 부종병은 Stx2e 독소를 생산하는 대장균(부종 병균)이 상부 장관 내에서 급격하게 증식하여 독소가 혈중에 흡수됨으로써 발병되는 세균성 질병으로, 4 내지 12 주령의 자돈에게 다발하여, 안검 부종이나 신경 증상 등을 나타내고, 그 대부분은 발병 후 24시간 이내에 폐사한다. 부종병균의 감염에 의한 치사율은 50 내지 90%에 이를 정도로 매우 높고, 재발이나 발육 불량 등에 의한 증체율 저하를 초래하여 경제적으로 매우 큰 손실을 주는 것으로 알려져 있다.It is reported that swine edema disease is mainly caused by Stx2e + Escherichia coli ( E.coli ). In particular, swine edema disease is a major bacterial disease causing enormous economic loss to pig farms following the recent Foot and Mouth Disease (FMD) and porcine epidemic diarrhea (PED). Pork edema disease produces Stx2e toxin. It is a bacterial disease that occurs when Escherichia coli (edema bacteria) rapidly proliferates in the upper intestinal tract and the toxin is absorbed into the blood. It occurs frequently in piglets aged 4 to 12 weeks, and causes eyelid edema and neurological symptoms, and most cases of this disease occur. They die within 24 hours. The mortality rate due to infection with edema pathogens is very high, reaching 50 to 90%, and it is known to cause a large economic loss by causing a decrease in weight gain due to recurrence or poor growth.
국내에서는 돼지 부종병 예방용 백신이 수입되어 제품이 상용화되어 있으나, 그 수입량이 많지 않고 가격이 고가이다. 현장에서의 수입 제품에 대한 평가는 엇갈리고 있는 실정으로, 저렴하면서 보다 우수한 효능을 가진 제품 개발을 요구하고 있는 실정이다. 또한 아직까지 수입 물량이 적어 필요로 하는 농장에 다 보급되지 못하고 있어, 질병 발생시 항생 물질로 치료하고 있지만, 발병 후 항생제 투여로는 치료시기를 놓치는 경우가 많다. 또한 다제 내성균의 출현도 보고되어 있어 유효한 예방법이나 치료법 개발이 절실히 요구되고 있다. In Korea, a vaccine for preventing swine edema disease has been imported and commercialized, but the quantity imported is not large and the price is high. The evaluation of imported products in the field is mixed, and there is a demand for the development of products that are less expensive and have better efficacy. In addition, the quantity imported is still small and cannot be supplied to all farms that need it, so when a disease occurs, it is treated with antibiotics, but treatment time is often missed when antibiotics are administered after the disease occurs. Additionally, the emergence of multi-drug resistant bacteria has been reported, so there is an urgent need to develop effective prevention methods or treatments.
Stx2e는 rRNA N-글리코시다아제 활성을 갖는 A subunit(Stx2eA)과 수용체(globotetraosyl ceramide(Gb4))와 결합하는 오량체의 B subunit(Stx2eB)으로 구성되는 전형적인 AB5형의 독소 단백질이다. 장관으로부터 흡수된 Stx2e는 오량체의 B subunit에 의해 혈관 내피세포 등의 세포와 결합하고 endosome 형태로 세포내로 흡수된 후 Stx2e의 A subunit이 리보솜의 단백질 합성을 저해하여 부종병을 일으키는 것으로 알려져 있다. 다만, 종래 개발된 Stx2e 기반의 부종병 예방용 재조합 단백질의 경우, 대량 발현에 부적합하며 순도가 낮아 주사용 백신용도로 적합하지 않다.Stx2e is a typical AB 5 type toxin protein composed of an A subunit (Stx2eA) with rRNA N-glycosidase activity and a pentameric B subunit (Stx2eB) that binds to a receptor (globotetraosyl ceramide (Gb4)). Stx2e absorbed from the intestinal tract is known to bind to cells such as vascular endothelial cells by the pentameric B subunit and to be absorbed into cells in the form of endosomes, and then the A subunit of Stx2e inhibits ribosomal protein synthesis, causing edema disease. However, the conventionally developed Stx2e-based recombinant protein for preventing edema disease is unsuitable for mass expression and has low purity, making it unsuitable for use as an injectable vaccine.
이에 본 발명자들은 대량생산이 가능하면서도 돼지 부종병의 예방 효과가 우수한 백신 개발을 위한 연구를 수행하여 본 발명을 완성하였다.Accordingly, the present inventors completed the present invention by conducting research to develop a vaccine that can be mass-produced and is excellent in preventing swine edema disease.
본 발명의 하나의 목적은 서열번호 1의 아미노산 서열 및 서열번호 2의 아미노산 서열을 포함하는 재조합 단백질을 제공하는 것이다.One object of the present invention is to provide a recombinant protein comprising the amino acid sequence of SEQ ID NO: 1 and the amino acid sequence of SEQ ID NO: 2.
본 발명의 다른 목적은 서열번호 3을 포함하는 제1 재조합 발현 벡터 및 서열번호 4를 포함하는 제2 재조합 발현 벡터로 대장균을 형질전환하는 단계; 형질전환된 대장균에서 제1 재조합 발현 벡터 및 제2 재조합 발현 벡터를 과발현시켜 재조합 단백질의 발현을 유도하는 단계; 및 발현된 재조합 단백질을 정제하는 단계를 포함하는 재조합 단백질의 대량생산 방법을 제공하는 것이다.Another object of the present invention is to transform E. coli with a first recombinant expression vector containing SEQ ID NO: 3 and a second recombinant expression vector containing SEQ ID NO: 4; Inducing expression of a recombinant protein by overexpressing the first recombinant expression vector and the second recombinant expression vector in transformed E. coli; and purifying the expressed recombinant protein.
본 발명의 또 다른 목적은 상기 재조합 단백질을 포함하는 돼지 부종병 백신 조성물을 제공하는 것이다.Another object of the present invention is to provide a porcine edema disease vaccine composition containing the above recombinant protein.
본 발명의 또 다른 목적은 상기 백신 조성물을 모돈 또는 자돈에 접종하는 단계를 포함하는 돼지 부종병에 대한 면역력을 증진시키는 방법을 제공하는 것이다.Another object of the present invention is to provide a method for enhancing immunity against swine edema disease, comprising the step of inoculating the vaccine composition into sow or piglet.
본 발명의 또 다른 목적은 상기 재조합 단백질을 포함하는 돼지 부종병 예방용 사료 첨가제를 제공하는 것이다.Another object of the present invention is to provide a feed additive for preventing swine edema disease containing the recombinant protein.
본 발명의 일 양상은 서열번호 1의 아미노산 서열 및 서열번호 2의 아미노산 서열을 포함하는 재조합 단백질을 제공한다.One aspect of the present invention provides a recombinant protein comprising the amino acid sequence of SEQ ID NO: 1 and the amino acid sequence of SEQ ID NO: 2.
돼지 부종병을 예방하기 위하여 개발된 종래의 Stx2eA-C-말단 단편-Stx2eB-오량체 재조합 단백질은 intein과 6-his를 포함하고 있어 백신으로 사용할 수 없는 구조적인 한계를 가지고 있으며, 대량 생산이나 순도(purity)가 떨어져 주사용 백신으로 사용하기에는 한계점을 가지고 있다. 본 발명의 재조합 단백질은 부종병 원인 독소인 Stx2eA-(Stx2eB)5의 기본 틀을 유지하면서 intein과 6-his 없이 독소부분이 제거된 순수 변형 재조합 Stx2e 단백질로써 대량 발현 및 정제가 가능하며, 종래 Stx2eA-C-말단 단편-Stx2eB-오량체 재조합 단백질(100㎍/㎖) 대비 적은 양(10㎍/㎖)으로도 병원성 대장균에 대해 효과적으로 면역반응을 유도할 수 있으므로, 돼지 부종병 예방을 위한 백신 조성물로써 효과적으로 활용될 수 있다. The conventional Stx2eA-C-terminal fragment-Stx2eB-pentamer recombinant protein developed to prevent swine edema disease contains intein and 6-his, so it has structural limitations that prevent it from being used as a vaccine, and it cannot be mass-produced or purified. It has limitations in using it as an injectable vaccine due to its low purity. The recombinant protein of the present invention is a pure modified recombinant Stx2e protein with the toxin portion removed without intein and 6-his while maintaining the basic framework of Stx2eA-(Stx2eB) 5 , the toxin that causes edema disease. It can be expressed and purified in large quantities, and can be expressed and purified in large quantities. -C-terminal fragment-Stx2eB-pentamer recombinant protein (100㎍/㎖) can effectively induce an immune response against pathogenic E. coli even at a small amount (10㎍/㎖), so a vaccine composition for preventing swine edema disease This can be used effectively.
본 발명의 일 구체예에 따르면, 상기 재조합 단백질은 돼지 부종병에 대해 면역반응을 유도하는 것일 수 있다.According to one embodiment of the present invention, the recombinant protein may induce an immune response against swine edema disease.
본 발명의 다른 양상은 서열번호 3을 포함하는 제1 재조합 발현 벡터 및 서열번호 4를 포함하는 제2 재조합 발현 벡터로 대장균을 형질전환하는 단계; 형질전환된 대장균에서 제1 재조합 발현 벡터 및 제2 재조합 발현 벡터를 과발현시켜 재조합 단백질의 발현을 유도하는 단계; 및 발현된 재조합 단백질을 정제하는 단계를 포함하는 재조합 단백질의 대량생산 방법을 제공한다.Another aspect of the present invention includes transforming E. coli with a first recombinant expression vector comprising SEQ ID NO: 3 and a second recombinant expression vector comprising SEQ ID NO: 4; Inducing expression of a recombinant protein by overexpressing the first recombinant expression vector and the second recombinant expression vector in transformed E. coli; and purifying the expressed recombinant protein.
본 발명의 일 구체예에 따르면, 상기 정제하는 단계는 Ni-NTi 컬럼으로 상기 발현된 재조합 단백질을 제1 정제하는 단계; TEV 프로테이나제(proteinase)를 처리하는 단계; 및 MBP 컬럼으로 제2 정제하는 단계를 포함하는 것일 수 있다.According to one embodiment of the present invention, the purifying step includes first purifying the expressed recombinant protein using a Ni-NTi column; Processing TEV proteinase; And it may include a second purification step using an MBP column.
Ni-NTi 컬럼을 사용한 제1 정제에 의하여 재조합 6xhis-MBP-TEV-HJP2-Stx2eB 단백질이 정제되고, TEV 프로테이나제의 처리에 의하여 재조합 HJP2-Stx2eB 단백질이 생성되며, MBP 컬럼을 사용한 제2 정제에 의하여 최종적으로 순수한 재조합 HJP2-Stx2eB 단백질의 정제가 가능하다.The recombinant 6xhis-MBP-TEV-HJP2-Stx2eB protein is purified by the first purification using a Ni-NTi column, the recombinant HJP2-Stx2eB protein is produced by treatment with TEV proteinase, and the second purification using the MBP column Through purification, it is finally possible to purify pure recombinant HJP2-Stx2eB protein.
본 발명의 일 구체예에 따르면, 상기 대장균은 BL21(DE3)일 수 있다.According to one embodiment of the present invention, the E. coli may be BL21(DE3).
본 발명의 또 다른 양상은 상기 재조합 단백질을 포함하는 돼지 부종병 백신 조성물을 제공한다.Another aspect of the present invention provides a porcine edema disease vaccine composition comprising the above recombinant protein.
본 발명에서 사용되는 용어, "백신"은 생체에 면역을 주는 항원을 함유한 생물학적인 제제로서, 감염증의 예방을 위하여 사람이나 동물에 주사하거나 경구 투여함으로써 생체에 면역이 생기게 하는 면역원 또는 항원성 물질을 말한다.As used in the present invention, the term "vaccine" is a biological agent containing an antigen that provides immunity to a living body, and is an immunogen or antigenic substance that creates immunity in a living body by injecting or orally administering it to humans or animals to prevent infectious diseases. says
본 발명의 백신 조성물은 안정제, 유화제, 수산화알루미늄, 인산알루미늄, pH 조정제, 계면활성제, 리포솜, 이스콤 (iscom) 보조제, 합성 글리코펩티드, 증량제, 카복시폴리메틸렌, 서브바이랄 (subviral) 입자 보조제, 콜레라 독소, N,N-디옥타데실-N',N'-비스(2-하이드록시에틸)-프로판디아민, 모노포스포릴 지질 A, 디메틸디옥타데실-암모늄 브로마이드 및 이의 혼합물로 구성된 군에서 선택된 어느 하나 이상의 제2 보조제를 추가로 함유할 수 있다.The vaccine composition of the present invention includes stabilizers, emulsifiers, aluminum hydroxide, aluminum phosphate, pH adjusters, surfactants, liposomes, iscom adjuvants, synthetic glycopeptides, extenders, carboxypolymethylene, subviral particle adjuvants, selected from the group consisting of cholera toxin, N,N-dioctadecyl-N',N'-bis(2-hydroxyethyl)-propanediamine, monophosphoryl lipid A, dimethyldioctadecyl-ammonium bromide and mixtures thereof. It may additionally contain one or more second auxiliaries.
본 발명의 백신 조성물은 각각 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구형 제형 및 드립 (drip) 또는 스프레이 등의 비강용 제형 그리고 멸균 주사용액의 형태로 제형화하여 사용될 수 있다. 제제화할 경우에는 보통 사용되는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제할 수 있다. 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 상기 레시틴 유사 유화제에 적어도 하나 이상의 부형제 예를 들면, 전분, 칼슘카보네이트(calcium carbonate), 수크로스 또는 락토오스, 젤라틴 등을 섞어 조제할 수 있다. 또한 단순한 부형제 이외에 마그네슘 스티레이트 탈크 같은 윤활제들도 사용할 수 있다. 경구 투여를 위한 액상제제로는 현탁제, 내용액제, 유제, 시럽제 등을 사용할 수 있으며, 흔히 사용되는 단순 희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수용성제, 현탁제, 유제, 동결건조제제가 포함된다. 비수용성제제, 현탁제로는 프로필렌글리콜(propylene glycol), 폴리 에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있으나, 이에 제한되지 않는다. 비강내 투여를 위한 제제에 적합한 침투제는 일반적으로 당업자에게 공지되어 있다. 그러한 적합한 제형물은 안정성과 순응도를 위해 바람직하게 무균, 등장 및 완충되도록 제형화된다. 비강내 투여를 위한 제제는 또한 정상적인 섬모 작용을 유지시키기 위해 점액 분비를 여러 측면에서 자극하도록 제조되며, 문헌(Remington's Pharmaceutical Science, 18th Ed., Mack Publishing Co., Easton, PA(1990))에 기술된 바와 같이, 바람직하게는 pH 5.5 내지 6.5를 유지하는 등장성의 완충된 제형일 수 있으며, 추가적으로 항미생물 방부제 및 적합한 약물 안정화제를 포함할 수 있다.The vaccine composition of the present invention can be administered in oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, nasal formulations such as drips or sprays, and sterile injection solutions according to conventional methods. It can be formulated and used in a form. When formulating, it can be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations contain the lecithin-like emulsifier and at least one or more excipients, such as starch, calcium carbonate, and sucrose. Alternatively, it can be prepared by mixing lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium styrate talc can also be used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives can be used. may be included. Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous preparations, suspensions, emulsions, and lyophilized preparations. Non-aqueous preparations and suspensions include, but are not limited to, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. Penetrants suitable for formulations for intranasal administration are generally known to those skilled in the art. Such suitable formulations are preferably formulated to be sterile, isotonic and buffered for stability and compliance. Formulations for intranasal administration are also formulated to stimulate mucus secretion in several ways to maintain normal ciliary action, and are described in Remington's Pharmaceutical Science, 18th Ed., Mack Publishing Co., Easton, PA (1990). As described above, it may preferably be an isotonic buffered formulation maintaining pH 5.5 to 6.5, and may additionally include an antimicrobial preservative and a suitable drug stabilizer.
본 발명의 일 구체예에 따르면, 상기 조성물은 수산화 알루미늄, GEL 01, GEL 02, IMS 1313(VG), IMS 1313(VG N), ISA 15A(VG), ISA 28R(VG), ISA 35(VG), ISA 201(VG), ISA 206(VG), ISA 660(VG), 기름속 수형 에멀젼(물-in-oil emulsion), 불완전 프로인트항원보조제(불완전한 Freund’s adjuvant), 백반(alum), 알루미늄 하이드록사이드(aluminum hydroxide), Toll-like 수용체 작용제(Toll-like receptor agonist), 면역자극성 올리고뉴클레오티드(Immunostimulatory oligonucleotide) 및 생물학적 보조제(biological adjuvant)로 이루어진 군으로부터 선택되는 어느 하나 이상의 보조제(adjuvant)를 더 포함할 수 있다.According to one embodiment of the present invention, the composition includes aluminum hydroxide, GEL 01, GEL 02, IMS 1313 (VG), IMS 1313 (VG N), ISA 15A (VG), ISA 28R (VG), ISA 35 (VG) ), ISA 201(VG), ISA 206(VG), ISA 660(VG), water-in-oil emulsion, incomplete Freund's adjuvant, alum, aluminum One or more adjuvants selected from the group consisting of aluminum hydroxide, Toll-like receptor agonist, immunostimulatory oligonucleotide, and biological adjuvant. More may be included.
본 발명의 백신 조성물은 담체 또는 보조제를 추가적으로 포함할 수 있다. 상기 보조제는 수산화 알루미늄, GEL 01, GEL 02, IMS 1313(VG), IMS 1313(VG N), ISA 15A(VG), ISA 28R(VG), ISA 35(VG), ISA 201(VG), ISA 206(VG), ISA 660(VG), 기름속 수형 에멀젼(water-in-oil emulsion), 불완전한 프로인트항원보강제(Freund's adjuvant), 백반(alum), 알루미늄 하이드록사이드, Toll-like 수용체 작용제(agonist), 면역자극성(Immunostimulatory) 올리고뉴크레오티드 또는 생물학적 보조제일 수 있고, 바람직하게는 IMS 1313(VG)일 수 있다. 상기 담체란 임의의 및 모든 용매, 분산 매질, 코팅제, 항원 보강제, 안정제, 희석제, 보존제, 항균제 및 항진균제, 등장성 작용제, 흡착 지연제 등을 포함한다. 백신용 조성물에 포함될 수 있는 담체, 부형제, 희석제로는 락토즈, 덱스트로스, 슈크로스, 솔비톨, 만니톨, 자일리톨, 말티톨, 전분, 글리세린, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘포스페이트, 칼슘실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로즈, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유 등일 수 있다.The vaccine composition of the present invention may additionally include a carrier or adjuvant. The auxiliary agent is aluminum hydroxide, GEL 01, GEL 02, IMS 1313 (VG), IMS 1313 (VG N), ISA 15A (VG), ISA 28R (VG), ISA 35 (VG), ISA 201 (VG), ISA 206(VG), ISA 660(VG), water-in-oil emulsion, incomplete Freund's adjuvant, alum, aluminum hydroxide, Toll-like receptor agonist ( It may be an agonist, an immunostimulatory oligonucleotide, or a biological adjuvant, and is preferably IMS 1313 (VG). The carrier includes any and all solvents, dispersion media, coating agents, adjuvants, stabilizers, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, adsorption retardants, and the like. Carriers, excipients, and diluents that may be included in the vaccine composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, maltitol, starch, glycerin, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, It may be cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.
본 발명의 일 구체예에 따르면, 상기 조성물은 부피 1㎖를 기준으로 상기 재조합 단백질을 0.1㎍ 내지 1000㎍ 포함할 수 있다.According to one embodiment of the present invention, the composition may contain 0.1 μg to 1000 μg of the recombinant protein based on 1 ml of volume.
본 발명의 일 구체예에 따르면, 상기 돼지는 모돈 또는 자돈일 수 있다.According to one embodiment of the present invention, the pig may be a sow or a piglet.
본 발명의 조성물은 목적하는 방법에 따라 경구 투여하거나 비경구 투여(예를 들어, 근육 내, 정맥 내, 피하, 복강 내 또는 국소에 적용)할 수 있으며, 바람직하게는 경구 또는 근육 내 투여할 수 있으나, 이에 한정되는 것은 아니다. 상기 백신 조성물은 자돈 또는 모돈에 접종할 수 있으나, 가장 바람직하게는 자돈에 접종하는 것일 수 있다. 또한 상기 조성물의 투여량은 돼지의 무게, 연령, 성별, 건강상태, 식이, 투여시간, 투여방법, 배설율 및 질환의 중증도 등에 따라 그 범위가 다양하다. The composition of the present invention can be administered orally or parenterally (e.g., intramuscularly, intravenously, subcutaneously, intraperitoneally, or topically) depending on the desired method, and is preferably administered orally or intramuscularly. However, it is not limited to this. The vaccine composition can be inoculated into piglets or sows, but is most preferably inoculated into piglets. In addition, the dosage of the composition varies depending on the pig's weight, age, gender, health condition, diet, administration time, administration method, excretion rate, and severity of disease.
본 발명의 재조합 단백질은 백신 조성물 부피 1㎖를 기준으로 0.1㎍ 내지 1000㎍ 포함되어 투여될 수 있으며, 바람직하게는 1㎍ 내지 100㎍, 가장 바람직하게는 10㎍ 포함되어 투여될 수 있다.The recombinant protein of the present invention can be administered in an amount of 0.1 μg to 1000 μg, preferably 1 μg to 100 μg, and most preferably 10 μg based on 1 ml of vaccine composition volume.
본 발명의 또 다른 양상은 상기 백신 조성물을 모돈 또는 자돈에 접종하는 단계를 포함하는 돼지 부종병에 대한 면역력을 증진시키는 방법을 제공한다.Another aspect of the present invention provides a method for enhancing immunity against swine edema disease, comprising the step of inoculating the vaccine composition into a sow or piglet.
예를 들어, 본 발명의 백신 조성물은 2주 간격으로 2회 접종되어 부종병에 대한 효과적인 면역성을 제공할 수 있다.For example, the vaccine composition of the present invention can be administered twice at two-week intervals to provide effective immunity against edema disease.
본 발명의 또 다른 양상은 상기 재조합 단백질을 포함하는 돼지 부종병 예방용 사료 첨가제를 제공한다.Another aspect of the present invention provides a feed additive for preventing swine edema disease comprising the recombinant protein.
본 발명의 사료 첨가제는 재조합 HJP2-Stx2eB 단백질을 원형 그대로 사용하거나 또는 추가적으로 가축에 허용되는 곡류 및 그 부산물 등의 공지된 담체, 안정제 등을 가할 수 있으며, 필요에 따라 구연산, 후말산, 아디픽산, 젖산, 사과산 등의 유기산이나 인산나트륨, 인산칼륨, 산성 피로인산염, 폴리인산염 (중합인산염) 등의 인산염이나, 폴리페놀, 카테킨, 알파-토코페롤, 로즈마리 추출물, 비타민 C, 녹차 추출물, 감초 추출물, 키토산, 탄닌산, 피틴산 등의 천연 항산화제, 항생물질, 항균제 및 기타의 첨가제 등을 가할 수도 있으며, 그 형상으로서는 분체, 과립, 펠릿, 현탁액 등의 적당한 상태일 수 있으며, 상기 사료첨가제를 공급하는 경우는 가축 등에 대하여 단독으로 또는 사료에 혼합하여 공급할 수 있다.The feed additive of the present invention can be made by using the recombinant HJP2-Stx2eB protein in its original form, or by adding known carriers and stabilizers such as grains and their by-products acceptable to livestock, and if necessary, citric acid, humalic acid, adipic acid, Organic acids such as lactic acid and malic acid, phosphates such as sodium phosphate, potassium phosphate, acid pyrophosphate, and polyphosphate (polymerized phosphate), polyphenol, catechin, alpha-tocopherol, rosemary extract, vitamin C, green tea extract, licorice extract, and chitosan. , natural antioxidants such as tannic acid and phytic acid, antibiotics, antibacterial agents, and other additives may be added, and the form may be in an appropriate state such as powder, granule, pellet, or suspension. In the case of supplying the above feed additive, It can be supplied to livestock, etc. alone or mixed with feed.
재조합 Stx2e 단백질, 이의 대량생산 방법 및 이를 포함하는 돼지 부종병 백신 조성물에 따르면, 높은 순도로 재조합 단백질의 대량생산이 가능하며, 재조합 단백질은 현저한 항체 역가를 유도하여 돼지에게 부종병에 대한 면역능을 부여할 수 있으므로, 돼지, 특히 자돈의 부종병 예방을 위한 백신 조성물로써 유용하게 활용될 수 있다.According to the recombinant Stx2e protein, its mass production method, and the porcine edema disease vaccine composition containing the same, mass production of the recombinant protein is possible with high purity, and the recombinant protein induces a significant antibody titer and confers immunity against edema disease to pigs. Therefore, it can be usefully used as a vaccine composition to prevent edema disease in pigs, especially piglets.
도 1은 재조합 6xhis-MBP-TEV-HJP2 단백질 발현 재조합 벡터 pMAL-c5X의 모식도이다.
도 2는 Stx2eB 단백질 발현 재조합 발현 pET28a의 모식도이다.
도 3은 본 발명의 일 구체예에 따른 재조합 HJP2-Stx2eB 단백질 발현 확인을 위한 SDS-PAGE 결과(M : Marker, TEV(-): TEV proteinase 처리 전, TEV(+): TEV proteinase 처리 후, inj- : 이온 교환 컬럼에 투여 전, FT : 이온 교환 컬럼에서 정제된 후)를 나타낸 사진이다.
도 4는 본 발명의 일 구체예에 따른 재조합 HJP2-Stx2eB 단백질을 포함하는 백신 조성물을 자돈에 접종한 후, 재조합 HJP2-Stx2eB 단백질에 대한 혈청 IgG 항체역가를 분석한 결과를 나타낸 그래프이다.Figure 1 is a schematic diagram of the recombinant 6xhis-MBP-TEV-HJP2 protein expression recombinant vector pMAL-c5X.
Figure 2 is a schematic diagram of recombinant pET28a expressing Stx2eB protein.
Figure 3 shows SDS-PAGE results for confirmation of recombinant HJP2-Stx2eB protein expression according to one embodiment of the present invention (M: Marker, TEV(-): before TEV proteinase treatment, TEV(+): after TEV proteinase treatment, inj -: This is a picture showing (before administration to the ion exchange column, FT: after purification in the ion exchange column).
Figure 4 is a graph showing the results of analyzing serum IgG antibody titers against the recombinant HJP2-Stx2eB protein after inoculating piglets with a vaccine composition containing the recombinant HJP2-Stx2eB protein according to an embodiment of the present invention.
이하 본 발명을 하나 이상의 실시예를 통하여 보다 상세하게 설명한다. 그러나, 이들 실시예는 본 발명을 예시적으로 설명하기 위한 것으로 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail through one or more examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.
실시예 1. 박테리아 균주, 플라스미드 및 균주 배양 조건Example 1. Bacterial strains, plasmids, and strain culture conditions
부종병 의심 자돈으로부터 분리된 야생형 Stx2e+ F18+ STEC 균주인 STEC150229 및 HJL573을 도전감염 균주로 준비하였으며, E.coli BL21(DE3)를 pMAL-c5x-6xhis-MBP-TEV-HJP2(도 1) 및 pET28a-Stx2eB(도 2) 플라스미드로 형질전환시켜 재조합 HJP2 균주를 제조하였다(표 1). HJP2는 Stx2eA의 213번 내지 319번째의 아미노산 단편이다. STEC150229, HJL573 및 HJP2 균주는 LB 액체배지(Luria-Bertani broth; Becton, Dickinson and Company, 미국) 또는 LB 한천배지에서 37℃로 배양되었다. STEC150229 and HJL573, wild-type Stx2e + F18 + STEC strains isolated from piglets suspected of edema disease, were prepared as challenge strains, and E.coli BL21(DE3) was used as pMAL-c5x-6xhis-MBP-TEV-HJP2 (Figure 1) and A recombinant HJP2 strain was prepared by transformation with the pET28a-Stx2eB (Figure 2) plasmid (Table 1). HJP2 is the 213th to 319th amino acid fragment of Stx2eA. STEC150229, HJL573, and HJP2 strains were cultured at 37°C in LB broth (Luria-Bertani broth; Becton, Dickinson and Company, USA) or LB agar medium.
실시예 2. 재조합 HJP2-Stx2eB 단백질의 발현 및 정제Example 2. Expression and purification of recombinant HJP2-Stx2eB protein
실시예 1에서 제작한 재조합 HJP2 균주를 암피실린(Ampicillin) 및 카나마이신(Kanamycin)이 포함된 100㎖의 LB 배지에 접종하여 37℃ 온도에서 200rpm 속도로 밤새 배양하였다. 그 후, 배양액 10㎖를 암피실린 및 카나마이신이 포함된 LB 배지 1L에 접종하여 O.D. 600 값이 0.5가 될 때까지 20℃ 온도에서 200rpm 속도로 배양하였다. IPTG(isopropyl β-D-1-thiogalactopyranoside)(Duchefa)를 최종농도 0.5mM이 되도록 첨가한 후 20℃ 온도에서 150rpm 속도로 16시간 동안 배양하여 재조합 6xhis-MBP-TEV-HJP2-Stx2eB 단백질의 과발현을 유도하였다. 배양된 세포들을 4℃, 5,000rpm에서 15분간 원심분리하여 농축한 다음 20㎖ 용균 버퍼(lysis buffer)(10mM Tris, 1M NaCl, pH8.0)로 재부유하고, French press 방법으로 파쇄하였다. 4℃에서 12,000rpm으로 25분간 원심분리하여 용출액 중 수용성 단백질을 농축하였고, AKTA prime FPLC system(GE healthcare, USA)에 연결되어 있는 Ni-NTi 컬럼을 이용하여 1차 정제한 다음, TEV 프로테이나제(proteinase)로 재조합 6xhis-MBP-TEV-HJP2-Stx2eB 단백질의 TEV를 절단하고, MBP 컬럼으로 2차 정제하여 순수한 재조합 HJP2-Stx2eB 단백질을 얻었다. The recombinant HJP2 strain prepared in Example 1 was inoculated into 100 ml of LB medium containing ampicillin and kanamycin and cultured overnight at 37°C at a speed of 200 rpm. Afterwards, 10 ml of culture medium was inoculated into 1 L of LB medium containing ampicillin and kanamycin and adjusted to O.D. It was cultured at a temperature of 20°C and a speed of 200rpm until the 600 value reached 0.5. IPTG (isopropyl β-D-1-thiogalactopyranoside) (Duchefa) was added to a final concentration of 0.5mM and then cultured for 16 hours at 20°C at a speed of 150rpm to overexpress the recombinant 6xhis-MBP-TEV-HJP2-Stx2eB protein. induced. The cultured cells were concentrated by centrifugation at 4°C and 5,000rpm for 15 minutes, then resuspended in 20ml lysis buffer (10mM Tris, 1M NaCl, pH8.0), and disrupted using the French press method. Water-soluble proteins in the eluate were concentrated by centrifugation at 12,000 rpm for 25 minutes at 4°C, and primary purification was performed using a Ni-NTi column connected to an AKTA prime FPLC system (GE healthcare, USA), followed by TEV protein TEV of the recombinant 6xhis-MBP-TEV-HJP2-Stx2eB protein was cleaved with proteinase and purified a second time using an MBP column to obtain pure recombinant HJP2-Stx2eB protein.
순수정제된 재조합 단백질을 15% SDS-PAGE로 분석하였으며, 정제된 표적(target) 단백질은 buffer C(PBS buffer, GE healthcare, USA)를 이용하여 투석한 다음 centrcon(cut-off 30kDa, Amicon, Millipore, Germany)을 이용하여 농축하였다. 그 후, 농축된 재조합 HJP2-Ste2eB 단백질의 농도를 단백질 정량법(Bradford protein assay)으로 측정하였다. 즉, 15% SDS-PAGE에 전기영동하여 정제한 재조합 단백질의 밴드를 확인한 결과, HJP2-Stx2eB에 해당하는 재조합 단백질 밴드가 관찰되었다(도 3). Purely purified recombinant proteins were analyzed by 15% SDS-PAGE, and the purified target proteins were dialyzed using buffer C (PBS buffer, GE healthcare, USA) and then centrcon (cut-off 30kDa, Amicon, Millipore). , Germany) was used to concentrate it. Afterwards, the concentration of the concentrated recombinant HJP2-Ste2eB protein was measured by protein quantification (Bradford protein assay). That is, as a result of confirming the band of the purified recombinant protein by electrophoresis on 15% SDS-PAGE, a recombinant protein band corresponding to HJP2-Stx2eB was observed (Figure 3).
실시예 3. 백신 접종 및 샘플 수집Example 3. Vaccination and sample collection
국내 전북 소재 종돈장에서 부종병 증상이 없고 Stx2e에 음성인 모돈 및 3일령 포유 자돈을 구입하였으며, 한국동물보호협회 지침에 따른 준수사항을 전북대 동물윤리위원회로부터 승인을 받아 실험에 사용하였다.Sows and 3-day-old suckling piglets with no symptoms of edema disease and negative for Stx2e were purchased from a breeding farm located in Jeonbuk, Korea, and were used in experiments with approval from the Chonbuk National University Animal Ethics Committee following the guidelines of the Korea Animal Protection Association.
구체적으로, 임신 모돈에 대해 통상적인 백신 프로그램에 따라 예방접종하였다. 예상되는 분만일 14일 전에 임신 모돈을 각각 자돈을 위한 열전등(heat lamp)과 분만틀을 가진 분만우리에 옮겼다. 임신 모돈은 항생제 또는 다른 생장 촉진제 없는 식단으로 급여하였다. 자돈은 출생 후 최대 21일까지 모돈과 함께 사육하였다. 전체 5마리 임신 모돈으로부터 출생한 자돈 40마리를 5그룹으로 나누었다. 자돈이 5일령이 되었을 때, 그룹 A, B, C, D 및 E로 나누고, 그룹 A 및 B 자돈에 대해서는 멸균 PBS 1㎖를 접종하였으며, 그룹 C, D 및 E 자돈에 대해서는 실시예 2에서 수득한 재조합 HJP2-Stx2eB 단백질을 IMS1313 adjuvant에 혼합하여 각각 5㎍/㎖, 10㎍/㎖ 및 20㎍/㎖ 용량으로 근육 접종하였으며(0 week post prime immunization; WPPI), 자돈이 19일령이 되었을 때 동일한 용량 및 방법으로 2차 접종하였다(2 WPPI). 자돈이 21일령이 되었을 때 전북대학교 특성화 캠퍼스로 각 그룹당 5마리씩 이송하여 1주일간의 적응기간을 보냈다. 혈액 샘플은 2차 접종 전(2 WPPI) 그리고 도전감염 전(5주령, 4 WPPI)에 각각 채혈하여 수집하였으며, 모든 샘플은 사용 전까지 -70℃에 저장하였다. Specifically, pregnant sows were vaccinated according to a routine vaccination program. Fourteen days before the expected farrowing date, pregnant sows were transferred to farrowing pens, each equipped with a heat lamp and farrowing frame for the piglets. Pregnant sows were fed a diet without antibiotics or other growth promoters. Piglets were raised with the sows for up to 21 days after birth. A total of 40 piglets born from 5 pregnant sows were divided into 5 groups. When the piglets were 5 days old, they were divided into groups A, B, C, D and E. Groups A and B piglets were inoculated with 1 ml of sterile PBS, and group C, D and E piglets were obtained in Example 2. One recombinant HJP2-Stx2eB protein was mixed with IMS1313 adjuvant and intramuscularly inoculated at doses of 5㎍/㎖, 10㎍/㎖, and 20㎍/㎖, respectively (0 week post prime immunization; WPPI), and the same injection was administered when the piglets were 19 days old. The second vaccination was administered by dose and method (2 WPPI). When the piglets were 21 days old, they were transported to the specialized campus of Chonbuk National University, five in each group, and spent one week adapting. Blood samples were collected before the second vaccination (2 WPPI) and before challenge infection (5 weeks of age, 4 WPPI), and all samples were stored at -70°C until use.
실시예 4. 재조합 HJP2-Stx2eB 단백질의 현저한 혈청 IgG 역가 증가 효과 확인Example 4. Confirmation of significant serum IgG titer increase effect of recombinant HJP2-Stx2eB protein
부종병에 대한 재조합 HJP2-Stx2eB 단백질의 면역증진 효과를 실험하기 위해, pig IgG ELISA Quantitation Kit(Bethyl Lab Inc., 미국)를 사용하여 효소면역분석법(ELISA)을 수행하였다. To test the immune-enhancing effect of the recombinant HJP2-Stx2eB protein on edema disease, enzyme-linked immunosorbent assay (ELISA) was performed using the pig IgG ELISA Quantitation Kit (Bethyl Lab Inc., USA).
구체적으로, 실시예 3에서 수집한 자돈의 혈청 샘플을 1:200으로 희석하여 IgG 역가 측정에 사용하였으며, o-페닐렌디아민(Sigma-Aldrich, 미국)으로 발색하여 492nm에서 흡광도를 측정하였다.Specifically, the piglet serum sample collected in Example 3 was diluted 1:200 and used to measure IgG titer, developed with o -phenylenediamine (Sigma-Aldrich, USA), and absorbance was measured at 492 nm.
그 결과, 재조합 HJP2-Stx2eB 단백질 항원에 대한 혈청 IgG 역가는 대조군인 그룹 A과 PBS를 접종한 그룹 B에서 유사하게 나타난 반면, 재조합 HJP2-Stx2eB 단백질 항원을 접종한 그룹 C 내지 E 모두에서 대조군 대비 현저하게 증가한 것으로 나타났다(P≤0.05)(도 4).As a result, the serum IgG titer against the recombinant HJP2-Stx2eB protein antigen was similar in group A, which was the control group, and group B, which was inoculated with PBS, while it was significantly higher in both groups C to E, which were inoculated with the recombinant HJP2-Stx2eB protein antigen, compared to the control group. It was found to increase significantly (P≤0.05) (Figure 4).
실시예 5. 재조합 HJP2-Stx2eB 단백질의 부종병 예방 효과 확인Example 5. Confirmation of edema disease prevention effect of recombinant HJP2-Stx2eB protein
도전감염 균주를 사용하여 재조합 HJP2-Stx2eB 단백질의 부종병 예방 효과를 분석하였다.The effect of the recombinant HJP2-Stx2eB protein on preventing edema disease was analyzed using challenge infection strains.
구체적으로, HJL573과 STEC150229를 도전감염 균주(challenge strain)로 준비하고, 자돈이 5주령이 되었을 때 각 균주를 1×109 CFU/㎖로 준비하여 동량씩 혼합한 후 그룹 A 자돈을 제외한 모든 자돈에 2㎖씩 경구로 접종하였으며, 접종 후 7일 동안 매일 자돈의 임상증상 및 폐사 여부를 모니터링하였다. 부종병 증상이 발견된 자돈에서 직장도말(rectal swab)로 검체를 채취하여 병원성 대장균을 분리하였고, 표 2의 프라이머를 이용하여 Stx2e+ F18+ 대장균 여부를 PCR 방법으로 확인하였다. Specifically, HJL573 and STEC150229 were prepared as challenge strains, and when the piglets were 5 weeks old, each strain was prepared at 1 × 10 9 CFU/ml, mixed in equal amounts, and then injected into all piglets except group A piglets. They were inoculated orally at 2 ml each, and clinical symptoms and death of piglets were monitored daily for 7 days after inoculation. A rectal swab sample was collected from a piglet in which symptoms of edema disease were found, and pathogenic E. coli was isolated, and the presence of Stx2e + F18 + E. coli was confirmed by PCR using the primers in Table 2.
그 결과, 대조군인 그룹 B의 자돈 5두 모두 부종병의 임상 증상을 보였으며, 그룹 C의 자돈 2두에서 부종병의 증상이 관찰된 반면, 그룹 D 및 E의 자돈은 도전감염 7일까지 임상증상 및 폐사가 관찰되지 않은 것으로 나타났다(표 3).As a result, all 5 piglets in Group B, which was the control group, showed clinical symptoms of edema disease, and 2 piglets in Group C showed symptoms of edema disease, while piglets in Groups D and E showed clinical symptoms until 7 days after challenge infection. It was found that no symptoms or death were observed (Table 3).
이와 같은 결과를 통하여, 본 발명의 재조합 HJP2-Stx2eB 단백질은 자돈의 부종병 백신에 효과적으로 활용될 수 있으며, 특히 10㎍/㎖ 이상의 용량에서 더욱 효과적인 것으로 확인되었다.Through these results, it was confirmed that the recombinant HJP2-Stx2eB protein of the present invention can be effectively used as a vaccine against edema disease in piglets, and is especially effective at doses of 10 μg/ml or more.
이제까지 본 발명에 대하여 그 실시예들을 중심으로 살펴보았다. 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자는 본 발명이 본 발명의 본질적인 특성에서 벗어나지 않는 범위에서 변형된 형태로 구현될 수 있음을 이해할 수 있을 것이다. 그러므로 개시된 실시예들은 한정적인 관점이 아니라 설명적인 관점에서 고려되어야 한다. 본 발명의 범위는 전술한 설명이 아니라 청구범위에 나타나 있으며, 그와 동등한 범위 내에 있는 모든 차이점은 본 발명에 포함된 것으로 해석되어야 할 것이다.So far, the present invention has been examined focusing on its embodiments. A person skilled in the art to which the present invention pertains will understand that the present invention may be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a restrictive perspective. The scope of the present invention is indicated in the claims, not the foregoing description, and all differences within the equivalent scope should be construed as being included in the present invention.
<110> INDUSTRIAL COOPERATION FOUNDATION JEONBUK NATIONAL UNIVERSITY <120> Recombinant Stx2e Protein, Mass Production Method thereof, and Swine Edema Disease Vaccine Composition comprising the same <130> DHP22-129 <160> 8 <170> KoPatentIn 3.0 <210> 1 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of TEV-HJP2 <400> 1 Glu Asn Leu Tyr Phe Gln Met Thr Pro Glu Asp Val Asp Leu Thr Leu 1 5 10 15 Asn Trp Gly Arg Ile Ser Asn Val Leu Pro Glu Tyr Arg Gly Glu Ala 20 25 30 Gly Val Arg Val Gly Arg Ile Ser Phe Asn Asn Ile Ser Ala Ile Leu 35 40 45 Gly Thr Val Ala Val Ile Leu Asn Cys His His Gln Gly Ala Arg Ser 50 55 60 Val Arg Ala Val Asn Glu Glu Ser Gln Pro Glu Cys Gln Ile Thr Gly 65 70 75 80 Asp Arg Pro Val Ile Lys Ile Asn Asn Thr Leu Trp Glu Ser Asn Thr 85 90 95 Ala Ala Ala Phe Leu Asn Arg Lys Ser Gln Ser Leu Tyr Thr Thr Gly 100 105 110 Glu <210> 2 <211> 86 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of Stx2eB <400> 2 Lys Lys Met Phe Ile Ala Val Leu Phe Ala Leu Val Ser Val Asn Ala 1 5 10 15 Met Ala Ala Asp Cys Ala Lys Gly Lys Ile Glu Phe Ser Lys Tyr Asn 20 25 30 Glu Asp Asn Thr Phe Thr Val Lys Val Ser Gly Arg Glu Tyr Trp Thr 35 40 45 Asn Arg Trp Asn Leu Gln Pro Leu Leu Gln Ser Ala Gln Leu Thr Gly 50 55 60 Met Thr Val Thr Ile Ile Ser Asn Thr Cys Ser Ser Gly Ser Gly Phe 65 70 75 80 Ala Gln Val Lys Phe Asn 85 <210> 3 <211> 342 <212> DNA <213> Artificial Sequence <220> <223> gene sequence for expressing TEV-HJP2 in plasmid <400> 3 gaaaacctgt atttccagat gaccccggaa gacgtggatc tgaccctgaa ctggggtcgt 60 attagcaacg ttctgccgga gtatcgtggc gaagcgggtg tgcgtgttgg ccgtatcagc 120 tttaacaaca tcagcgcgat tctgggcacc gtggcggtta ttctgaactg ccatcaccaa 180 ggcgcgcgta gcgtgcgtgc ggttaacgag gaaagccagc cggagtgcca aattaccggc 240 gatcgtccgg tgatcaagat taacaacacc ctgtgggaaa gcaataccgc ggcggcgttt 300 ctgaatcgta agagccaaag cctgtacacc accggcgagt aa 342 <210> 4 <211> 282 <212> DNA <213> Artificial Sequence <220> <223> gene sequence for expressing Stx2eB in plasmid <400> 4 catatgaaga agatgtttat agcggtttta tttgcattgg tttctgttaa tgcaatggcg 60 gcggattgtg ctaaaggtaa aattgagttt tccaagtata atgaggataa tacctttact 120 gtgaaggtgt caggaagaga atactggacg aacagatgga atttgcagcc attgttacaa 180 agtgctcagc tgacagggat gactgtaaca atcatatcta atacctgcag ttcaggctca 240 ggctttgccc aggtgaagtt taactgagcg gccgcactcg ag 282 <210> 5 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> F18_forward <400> 5 tggcactgta ggagatacca ttcagc 26 <210> 6 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> F18_reverse <400> 6 ggtttgacca cctttcagtt gagcag 26 <210> 7 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Stx2e_forward <400> 7 cggtatccta ttcccaggag tttacg 26 <210> 8 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Stx2e_reverse <400> 8 gtcttccggc gtcatcgtat aaacag 26 <110> INDUSTRIAL COOPERATION FOUNDATION JEONBUK NATIONAL UNIVERSITY <120> Recombinant Stx2e Protein, Mass Production Method thereof, and Swine Edema Disease Vaccine Composition comprising the same <130>DHP22-129 <160> 8 <170> KoPatentIn 3.0 <210> 1 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of TEV-HJP2 <400> 1 Glu Asn Leu Tyr Phe Gln Met Thr Pro Glu Asp Val Asp Leu Thr Leu 1 5 10 15 Asn Trp Gly Arg Ile Ser Asn Val Leu Pro Glu Tyr Arg Gly Glu Ala 20 25 30 Gly Val Arg Val Gly Arg Ile Ser Phe Asn Asn Ile Ser Ala Ile Leu 35 40 45 Gly Thr Val Ala Val Ile Leu Asn Cys His His Gln Gly Ala Arg Ser 50 55 60 Val Arg Ala Val Asn Glu Glu Ser Gln Pro Glu Cys Gln Ile Thr Gly 65 70 75 80 Asp Arg Pro Val Ile Lys Ile Asn Asn Thr Leu Trp Glu Ser Asn Thr 85 90 95 Ala Ala Ala Phe Leu Asn Arg Lys Ser Gln Ser Leu Tyr Thr Thr Gly 100 105 110 Glu <210> 2 <211> 86 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of Stx2eB <400> 2 Lys Lys Met Phe Ile Ala Val Leu Phe Ala Leu Val Ser Val Asn Ala 1 5 10 15 Met Ala Ala Asp Cys Ala Lys Gly Lys Ile Glu Phe Ser Lys Tyr Asn 20 25 30 Glu Asp Asn Thr Phe Thr Val Lys Val Ser Gly Arg Glu Tyr Trp Thr 35 40 45 Asn Arg Trp Asn Leu Gln Pro Leu Leu Gln Ser Ala Gln Leu Thr Gly 50 55 60 Met Thr Val Thr Ile Ile Ser Asn Thr Cys Ser Ser Gly Ser Gly Phe 65 70 75 80 Ala Gln Val Lys Phe Asn 85 <210> 3 <211> 342 <212> DNA <213> Artificial Sequence <220> <223> gene sequence for expressing TEV-HJP2 in plasmid <400> 3 gaaaacctgt atttccagat gaccccggaa gacgtggatc tgaccctgaa ctggggtcgt 60 attagcaacg ttctgccgga gtatcgtggc gaagcgggtg tgcgtgttgg ccgtatcagc 120 tttaacaaca tcagcgcgat tctgggcacc gtggcggtta ttctgaactg ccatcaccaa 180 ggcgcgcgta gcgtgcgtgc ggttaacgag gaaagccagc cggagtgcca aattaccggc 240 gatcgtccgg tgatcaagat taacaacacc ctgtgggaaa gcaataccgc ggcggcgttt 300 ctgaatcgta agagccaaag cctgtacacc accggcgagt aa 342 <210> 4 <211> 282 <212> DNA <213> Artificial Sequence <220> <223> gene sequence for expressing Stx2eB in plasmid <400> 4 catatgaaga agatgtttat agcggtttta tttgcattgg tttctgttaa tgcaatggcg 60 gcggattgtg ctaaaggtaa aattgagttt tccaagtata atgaggataa tacctttact 120 gtgaaggtgt caggaagaga atactggacg aacagatgga atttgcagcc attgttacaa 180 agtgctcagc tgacagggat gactgtaaca atcatatcta atacctgcag ttcaggctca 240 ggctttgccc aggtgaagtt taactgagcg gccgcactcg ag 282 <210> 5 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> F18_forward <400> 5 tggcactgta ggagatacca ttcagc 26 <210> 6 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> F18_reverse <400> 6 ggtttgacca cctttcagtt gagcag 26 <210> 7 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Stx2e_forward <400> 7 cggtatccta ttcccaggag tttacg 26 <210> 8 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Stx2e_reverse <400> 8 gtcttccggc gtcatcgtat aaacag 26
Claims (11)
A recombinant protein comprising the amino acid sequence of SEQ ID NO: 1 and the amino acid sequence of SEQ ID NO: 2.
The recombinant protein according to claim 1, wherein the recombinant protein induces an immune response against swine edema disease.
형질전환된 대장균에서 제1 재조합 발현 벡터 및 제2 재조합 발현 벡터를 과발현시켜 재조합 단백질의 발현을 유도하는 단계; 및
발현된 재조합 단백질을 정제하는 단계
를 포함하는 재조합 단백질의 대량생산 방법.
Transforming E. coli with a first recombinant expression vector containing SEQ ID NO: 3 and a second recombinant expression vector containing SEQ ID NO: 4;
Inducing expression of a recombinant protein by overexpressing the first recombinant expression vector and the second recombinant expression vector in transformed E. coli; and
Purifying the expressed recombinant protein
Method for mass production of recombinant protein comprising.
Ni-NTi 컬럼으로 상기 발현된 재조합 단백질을 제1 정제하는 단계;
TEV 프로테이나제(proteinase)를 처리하는 단계; 및
MBP 컬럼으로 제2 정제하는 단계
를 포함하는 것인 재조합 단백질의 대량생산 방법.
The method of claim 3, wherein the purifying step is
First purifying the expressed recombinant protein using a Ni-NTi column;
Processing TEV proteinase; and
Second purification step with MBP column
A method for mass production of a recombinant protein comprising.
The method of mass producing a recombinant protein according to claim 3, wherein the E. coli strain is BL21(DE3).
A porcine edema disease vaccine composition comprising the recombinant protein of claim 1.
The vaccine composition according to claim 6, wherein the pig is a sow or piglet.
7. The method of claim 6, wherein the composition comprises aluminum hydroxide, GEL 01, GEL 02, IMS 1313 (VG), IMS 1313 (VG N), ISA 15A (VG), ISA 28R (VG), ISA 35 (VG), ISA 201(VG), ISA 206(VG), ISA 660(VG), water-in-oil emulsion, incomplete Freund's adjuvant, alum, aluminum hydroxide Further comprising one or more adjuvants selected from the group consisting of (aluminum hydroxide), Toll-like receptor agonist, immunostimulatory oligonucleotide, and biological adjuvant. A vaccine composition.
The vaccine composition according to claim 6, wherein the composition contains 0.1 μg to 1000 μg of the recombinant protein based on 1 ml of volume.
A method for enhancing immunity against swine edema disease, comprising the step of inoculating the vaccine composition of claim 6 into sow or piglet.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020220042625A KR20230143693A (en) | 2022-04-06 | 2022-04-06 | Recombinant Stx2e Protein, Mass Production Method thereof, and Swine Edema Disease Vaccine Composition comprising the same |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020220042625A KR20230143693A (en) | 2022-04-06 | 2022-04-06 | Recombinant Stx2e Protein, Mass Production Method thereof, and Swine Edema Disease Vaccine Composition comprising the same |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| KR20230143693A true KR20230143693A (en) | 2023-10-13 |
Family
ID=88289852
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| KR1020220042625A KR20230143693A (en) | 2022-04-06 | 2022-04-06 | Recombinant Stx2e Protein, Mass Production Method thereof, and Swine Edema Disease Vaccine Composition comprising the same |
Country Status (1)
| Country | Link |
|---|---|
| KR (1) | KR20230143693A (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20160133120A (en) | 2015-05-12 | 2016-11-22 | (주)애드바이오텍 | Feed or feed additive with IgY for prevention of edema disease of swine |
-
2022
- 2022-04-06 KR KR1020220042625A patent/KR20230143693A/en unknown
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20160133120A (en) | 2015-05-12 | 2016-11-22 | (주)애드바이오텍 | Feed or feed additive with IgY for prevention of edema disease of swine |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP2178558B1 (en) | Yersinia pestis antigens, vaccine compositions, and related methods | |
| US8900594B2 (en) | Oral recombinant Helicobacter pylori vaccine and preparing method thereof | |
| EP3319630A1 (en) | Vaccine against s.suis infection | |
| KR102103013B1 (en) | Vaccine composition for preventing or treating pathogenic Escherichia coli in pigs comprising Stx2eA-C-terminal fragment-(Stx2eB)5 recombinant protein, and F4+ enterotoxigenic E. coli and F18+ enterotoxigenic E. coli inactivated cells as a vaccine | |
| KR101818901B1 (en) | Inactivated vaccine composition of Actinobacillus pleuroneumoniae inactivated bacterin and N or C fragments of Actinobacillus pleuroneumniae ApxIA, ApxIIA, ApxIIIA inactivated toxin | |
| KR20110022428A (en) | Attenuated salmonella mutants transformed with adhesin gene of pathogenic escherichia coli in pigs and vaccine composition comprising thereof for protection and treatment against pathogenic escherichia coli and salmonella in pigs | |
| JP2012521391A (en) | Vaccines to protect against various serotypes of Streptococcus bacterium | |
| US20240131134A1 (en) | Periodontitis vaccine and related compositions and methods of use | |
| EP1657248B1 (en) | Vaccine for preventing and treating porcine progressive atrophic rhinitis | |
| EP2916863B1 (en) | Attenuated mannheimia haemolytica vaccines and methods of making and use | |
| KR101743442B1 (en) | Vaccine composition for preventing or treating porcine edema disease comprising ghost Salmonella mutant expressing Enterotoxigenic Escherichia coli antigen as effective component | |
| US8067203B2 (en) | Composition for treating porcine progressive atrophic rhinitis and making process thereof | |
| KR102018707B1 (en) | Vaccine composition for preventing or treating porcine pleuropneumonia | |
| KR20230143693A (en) | Recombinant Stx2e Protein, Mass Production Method thereof, and Swine Edema Disease Vaccine Composition comprising the same | |
| KR102439333B1 (en) | Vaccine composition for preventing or treating pathogenic Escherichia coli in pigs comprising Stx2eA2-(Stx2eB)5 recombinant protein as a vaccine | |
| KR102514051B1 (en) | Vaccine composition of recombinant Apx IA-C-terminal, Apx IIA-C-terminal, Apx IIIA-C-terminal and Apx IV-C-terminal toxoids | |
| KR20230119818A (en) | Recombinant Stx2e Protein, Swine Edema Disease Vaccine Composition comprising the same and Oral Dosage Form | |
| KR102100503B1 (en) | Vaccin compsosition for preventing or treating porcine pathogenic colibacillosis comprising ghost enterotoxigenic Escherichia coli as effective componet | |
| KR102249189B1 (en) | Vaccine composition for preventing or treating porcine proliferative enteritis and salmonellosis simultaneouly comprising attenuated Salmonella mutant expressing immunogen having improved antigenicity as effective component | |
| KR101950931B1 (en) | ptsI and crr mutated and attenuated Salmonella typhimurium and a composition for vaccine using the same | |
| JP7349366B2 (en) | Vaccines containing clostridial toxins | |
| KR101712012B1 (en) | Vaccine composition for preventing or treating porcine pathogenic colibacillosis comprising improved ghost Salmonella mutant as effective component | |
| Moon et al. | Efficacy evaluation of combination vaccine of recombinant C-terminal fragments of ApxIA, ApxIIA and ApxIIIA in piglets | |
| KR20220109129A (en) | Vaccine composition containing novel type of inactivated Salmonella Enteritidis whole cells for preventing or treating salmonellosis | |
| CN117120081A (en) | Immunogenic fusion proteins |