KR20190059652A - Protein comprising ectodomain of japanese encephalitis virus envelope proteins, homodimer thereof, and use thereof - Google Patents
Protein comprising ectodomain of japanese encephalitis virus envelope proteins, homodimer thereof, and use thereof Download PDFInfo
- Publication number
- KR20190059652A KR20190059652A KR1020170157475A KR20170157475A KR20190059652A KR 20190059652 A KR20190059652 A KR 20190059652A KR 1020170157475 A KR1020170157475 A KR 1020170157475A KR 20170157475 A KR20170157475 A KR 20170157475A KR 20190059652 A KR20190059652 A KR 20190059652A
- Authority
- KR
- South Korea
- Prior art keywords
- japanese encephalitis
- protein
- encephalitis virus
- seq
- antibody
- Prior art date
Links
- 241000710842 Japanese encephalitis virus Species 0.000 title claims abstract description 119
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 57
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 55
- 108010003533 Viral Envelope Proteins Proteins 0.000 title abstract description 13
- 239000000710 homodimer Substances 0.000 title abstract description 11
- 102000036639 antigens Human genes 0.000 claims abstract description 49
- 108091007433 antigens Proteins 0.000 claims abstract description 49
- 239000000427 antigen Substances 0.000 claims abstract description 31
- 229960005486 vaccine Drugs 0.000 claims abstract description 22
- 230000009385 viral infection Effects 0.000 claims abstract description 22
- 241000238631 Hexapoda Species 0.000 claims abstract description 14
- 238000003745 diagnosis Methods 0.000 claims abstract description 11
- 201000010099 disease Diseases 0.000 claims abstract description 11
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 11
- 239000004480 active ingredient Substances 0.000 claims abstract description 3
- 108700029642 Dicentrarchus labrax encephalitis virus Diev coat Proteins 0.000 claims description 55
- 210000004027 cell Anatomy 0.000 claims description 29
- 238000000034 method Methods 0.000 claims description 26
- 238000012360 testing method Methods 0.000 claims description 15
- 239000000178 monomer Substances 0.000 claims description 11
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 11
- 239000013604 expression vector Substances 0.000 claims description 9
- 238000010171 animal model Methods 0.000 claims description 6
- 239000002773 nucleotide Substances 0.000 claims description 6
- 125000003729 nucleotide group Chemical group 0.000 claims description 6
- 239000012634 fragment Substances 0.000 claims description 5
- 238000004519 manufacturing process Methods 0.000 claims description 5
- 241000701447 unidentified baculovirus Species 0.000 claims description 5
- 241001465754 Metazoa Species 0.000 claims description 4
- 206010014599 encephalitis Diseases 0.000 claims description 4
- 210000003527 eukaryotic cell Anatomy 0.000 claims description 4
- 230000028993 immune response Effects 0.000 claims description 4
- 229920001184 polypeptide Polymers 0.000 claims description 4
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 4
- 206010019233 Headaches Diseases 0.000 claims description 3
- 231100000869 headache Toxicity 0.000 claims description 3
- 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 2
- 208000006740 Aseptic Meningitis Diseases 0.000 claims description 2
- 206010027201 Meningitis aseptic Diseases 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims 2
- 239000012528 membrane Substances 0.000 claims 1
- 241000700605 Viruses Species 0.000 abstract description 18
- 206010014596 Encephalitis Japanese B Diseases 0.000 abstract description 13
- 201000005807 Japanese encephalitis Diseases 0.000 abstract description 13
- 230000014509 gene expression Effects 0.000 abstract description 13
- 239000008194 pharmaceutical composition Substances 0.000 abstract description 3
- 229940043026 inactivated japanese encephalitis virus vaccine Drugs 0.000 abstract 1
- 235000018102 proteins Nutrition 0.000 description 45
- 150000001413 amino acids Chemical group 0.000 description 22
- 210000002966 serum Anatomy 0.000 description 18
- 235000001014 amino acid Nutrition 0.000 description 15
- 229940024606 amino acid Drugs 0.000 description 15
- 238000002965 ELISA Methods 0.000 description 14
- 241000699666 Mus <mouse, genus> Species 0.000 description 10
- 239000013598 vector Substances 0.000 description 9
- 241000699670 Mus sp. Species 0.000 description 8
- 230000015572 biosynthetic process Effects 0.000 description 8
- 238000002474 experimental method Methods 0.000 description 8
- 108020004414 DNA Proteins 0.000 description 7
- 241000588724 Escherichia coli Species 0.000 description 7
- 210000004369 blood Anatomy 0.000 description 7
- 239000008280 blood Substances 0.000 description 7
- 241000710831 Flavivirus Species 0.000 description 6
- 108010052285 Membrane Proteins Proteins 0.000 description 6
- 102000018697 Membrane Proteins Human genes 0.000 description 6
- 101710132601 Capsid protein Proteins 0.000 description 5
- 101710094648 Coat protein Proteins 0.000 description 5
- 102100021181 Golgi phosphoprotein 3 Human genes 0.000 description 5
- 101710125418 Major capsid protein Proteins 0.000 description 5
- 101710141454 Nucleoprotein Proteins 0.000 description 5
- 101710083689 Probable capsid protein Proteins 0.000 description 5
- 238000001042 affinity chromatography Methods 0.000 description 5
- 230000003053 immunization Effects 0.000 description 5
- 238000000746 purification Methods 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 241000710781 Flaviviridae Species 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- 238000001641 gel filtration chromatography Methods 0.000 description 4
- 229940031551 inactivated vaccine Drugs 0.000 description 4
- 230000003472 neutralizing effect Effects 0.000 description 4
- 239000002953 phosphate buffered saline Substances 0.000 description 4
- 239000000523 sample Substances 0.000 description 4
- 230000035945 sensitivity Effects 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 241000714197 Avian myeloblastosis-associated virus Species 0.000 description 3
- 108010049041 glutamylalanine Proteins 0.000 description 3
- VPZXBVLAVMBEQI-UHFFFAOYSA-N glycyl-DL-alpha-alanine Natural products OC(=O)C(C)NC(=O)CN VPZXBVLAVMBEQI-UHFFFAOYSA-N 0.000 description 3
- XKUKSGPZAADMRA-UHFFFAOYSA-N glycyl-glycyl-glycine Chemical compound NCC(=O)NCC(=O)NCC(O)=O XKUKSGPZAADMRA-UHFFFAOYSA-N 0.000 description 3
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 3
- 238000002649 immunization Methods 0.000 description 3
- 230000016784 immunoglobulin production Effects 0.000 description 3
- 208000015181 infectious disease Diseases 0.000 description 3
- 238000011081 inoculation Methods 0.000 description 3
- 230000002018 overexpression Effects 0.000 description 3
- 230000002265 prevention Effects 0.000 description 3
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 3
- 238000010998 test method Methods 0.000 description 3
- JDIQCVUDDFENPU-ZKWXMUAHSA-N Ala-His-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@@H](N)C)CC1=CNC=N1 JDIQCVUDDFENPU-ZKWXMUAHSA-N 0.000 description 2
- BLTRAARCJYVJKV-QEJZJMRPSA-N Ala-Lys-Phe Chemical compound C[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](Cc1ccccc1)C(O)=O BLTRAARCJYVJKV-QEJZJMRPSA-N 0.000 description 2
- 108090000565 Capsid Proteins Proteins 0.000 description 2
- 102000009016 Cholera Toxin Human genes 0.000 description 2
- 108010049048 Cholera Toxin Proteins 0.000 description 2
- 238000009007 Diagnostic Kit Methods 0.000 description 2
- 101710091045 Envelope protein Proteins 0.000 description 2
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 2
- LCNXZQROPKFGQK-WHFBIAKZSA-N Gly-Asp-Ser Chemical compound NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(O)=O LCNXZQROPKFGQK-WHFBIAKZSA-N 0.000 description 2
- BDFCIKANUNMFGB-PMVVWTBXSA-N His-Gly-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CC1=CN=CN1 BDFCIKANUNMFGB-PMVVWTBXSA-N 0.000 description 2
- 102100034349 Integrase Human genes 0.000 description 2
- 229940124868 Japanese encephalitis virus vaccine Drugs 0.000 description 2
- WNGVUZWBXZKQES-YUMQZZPRSA-N Leu-Ala-Gly Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C)C(=O)NCC(O)=O WNGVUZWBXZKQES-YUMQZZPRSA-N 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- 101710188315 Protein X Proteins 0.000 description 2
- 206010057190 Respiratory tract infections Diseases 0.000 description 2
- HSWXBJCBYSWBPT-GUBZILKMSA-N Ser-Val-Val Chemical compound CC(C)[C@H](NC(=O)[C@@H](NC(=O)[C@@H](N)CO)C(C)C)C(O)=O HSWXBJCBYSWBPT-GUBZILKMSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- CJXURNZYNHCYFD-WDCWCFNPSA-N Thr-Lys-Gln Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N)O CJXURNZYNHCYFD-WDCWCFNPSA-N 0.000 description 2
- LYERIXUFCYVFFX-GVXVVHGQSA-N Val-Leu-Glu Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)O)NC(=O)[C@H](C(C)C)N LYERIXUFCYVFFX-GVXVVHGQSA-N 0.000 description 2
- 210000005013 brain tissue Anatomy 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 238000010835 comparative analysis Methods 0.000 description 2
- 238000000502 dialysis Methods 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 108010078144 glutaminyl-glycine Proteins 0.000 description 2
- 108010019832 glycyl-asparaginyl-glycine Proteins 0.000 description 2
- 108010026364 glycyl-glycyl-leucine Proteins 0.000 description 2
- 108010050848 glycylleucine Proteins 0.000 description 2
- 230000036039 immunity Effects 0.000 description 2
- 230000000091 immunopotentiator Effects 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 238000007912 intraperitoneal administration Methods 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 238000000691 measurement method Methods 0.000 description 2
- 238000006386 neutralization reaction Methods 0.000 description 2
- 150000007523 nucleic acids Chemical group 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 235000020183 skimmed milk Nutrition 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 108010061238 threonyl-glycine Proteins 0.000 description 2
- 239000012096 transfection reagent Substances 0.000 description 2
- 230000003612 virological effect Effects 0.000 description 2
- 238000001262 western blot Methods 0.000 description 2
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 1
- SEFVRKXJJPMVHQ-YUMQZZPRSA-N (2s)-2-[[2-[[(2s)-2-[(2-aminoacetyl)amino]-5-(diaminomethylideneamino)pentanoyl]amino]acetyl]amino]butanedioic acid Chemical compound NC(N)=NCCC[C@H](NC(=O)CN)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(O)=O SEFVRKXJJPMVHQ-YUMQZZPRSA-N 0.000 description 1
- FMYBFLOWKQRBST-UHFFFAOYSA-N 2-[bis(carboxymethyl)amino]acetic acid;nickel Chemical compound [Ni].OC(=O)CN(CC(O)=O)CC(O)=O FMYBFLOWKQRBST-UHFFFAOYSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- WRDANSJTFOHBPI-FXQIFTODSA-N Ala-Arg-Cys Chemical compound C[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CS)C(=O)O)N WRDANSJTFOHBPI-FXQIFTODSA-N 0.000 description 1
- BUDNAJYVCUHLSV-ZLUOBGJFSA-N Ala-Asp-Ser Chemical compound C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(O)=O BUDNAJYVCUHLSV-ZLUOBGJFSA-N 0.000 description 1
- BTYTYHBSJKQBQA-GCJQMDKQSA-N Ala-Asp-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](C)N)O BTYTYHBSJKQBQA-GCJQMDKQSA-N 0.000 description 1
- NBTGEURICRTMGL-WHFBIAKZSA-N Ala-Gly-Ser Chemical compound C[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O NBTGEURICRTMGL-WHFBIAKZSA-N 0.000 description 1
- LNNSWWRRYJLGNI-NAKRPEOUSA-N Ala-Ile-Val Chemical compound C[C@H](N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C(C)C)C(O)=O LNNSWWRRYJLGNI-NAKRPEOUSA-N 0.000 description 1
- DXTYEWAQOXYRHZ-KKXDTOCCSA-N Ala-Phe-Tyr Chemical compound C[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC2=CC=C(C=C2)O)C(=O)O)N DXTYEWAQOXYRHZ-KKXDTOCCSA-N 0.000 description 1
- RTZCUEHYUQZIDE-WHFBIAKZSA-N Ala-Ser-Gly Chemical compound C[C@H](N)C(=O)N[C@@H](CO)C(=O)NCC(O)=O RTZCUEHYUQZIDE-WHFBIAKZSA-N 0.000 description 1
- ARHJJAAWNWOACN-FXQIFTODSA-N Ala-Ser-Val Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(O)=O ARHJJAAWNWOACN-FXQIFTODSA-N 0.000 description 1
- LTTLSZVJTDSACD-OWLDWWDNSA-N Ala-Thr-Trp Chemical compound [H]N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O LTTLSZVJTDSACD-OWLDWWDNSA-N 0.000 description 1
- AETQNIIFKCMVHP-UVBJJODRSA-N Ala-Trp-Arg Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O AETQNIIFKCMVHP-UVBJJODRSA-N 0.000 description 1
- ZCUFMRIQCPNOHZ-NRPADANISA-N Ala-Val-Gln Chemical compound C[C@@H](C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N ZCUFMRIQCPNOHZ-NRPADANISA-N 0.000 description 1
- AFNHFVVOJZBIJD-GUBZILKMSA-N Arg-Met-Asp Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(O)=O AFNHFVVOJZBIJD-GUBZILKMSA-N 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- HOIFSHOLNKQCSA-FXQIFTODSA-N Asn-Arg-Asp Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(O)=O HOIFSHOLNKQCSA-FXQIFTODSA-N 0.000 description 1
- BDMIFVIWCNLDCT-CIUDSAMLSA-N Asn-Arg-Glu Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(O)=O BDMIFVIWCNLDCT-CIUDSAMLSA-N 0.000 description 1
- QISZHYWZHJRDAO-CIUDSAMLSA-N Asn-Asp-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CC(=O)N)N QISZHYWZHJRDAO-CIUDSAMLSA-N 0.000 description 1
- ZPMNECSEJXXNBE-CIUDSAMLSA-N Asn-Cys-Leu Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(C)C)C(O)=O ZPMNECSEJXXNBE-CIUDSAMLSA-N 0.000 description 1
- PLVAAIPKSGUXDV-WHFBIAKZSA-N Asn-Gly-Cys Chemical compound C([C@@H](C(=O)NCC(=O)N[C@@H](CS)C(=O)O)N)C(=O)N PLVAAIPKSGUXDV-WHFBIAKZSA-N 0.000 description 1
- ZTRJUKDEALVRMW-SRVKXCTJSA-N Asn-His-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)N[C@@H](CC2=CN=CN2)C(=O)O)NC(=O)[C@H](CC(=O)N)N ZTRJUKDEALVRMW-SRVKXCTJSA-N 0.000 description 1
- XVBDDUPJVQXDSI-PEFMBERDSA-N Asn-Ile-Glu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)O)NC(=O)[C@H](CC(=O)N)N XVBDDUPJVQXDSI-PEFMBERDSA-N 0.000 description 1
- ZYPWIUFLYMQZBS-SRVKXCTJSA-N Asn-Lys-Lys Chemical compound C(CCN)C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CC(=O)N)N ZYPWIUFLYMQZBS-SRVKXCTJSA-N 0.000 description 1
- UGXYFDQFLVCDFC-CIUDSAMLSA-N Asn-Ser-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O UGXYFDQFLVCDFC-CIUDSAMLSA-N 0.000 description 1
- ZUFPUBYQYWCMDB-NUMRIWBASA-N Asn-Thr-Glu Chemical compound NC(=O)C[C@H](N)C(=O)N[C@@H]([C@H](O)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O ZUFPUBYQYWCMDB-NUMRIWBASA-N 0.000 description 1
- AXXCUABIFZPKPM-BQBZGAKWSA-N Asp-Arg-Gly Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(O)=O AXXCUABIFZPKPM-BQBZGAKWSA-N 0.000 description 1
- SHBKFJNZNSGHDS-FGPLHTHASA-N Asp-Met-Thr-Pro Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](CCSC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N1CCC[C@H]1C(O)=O SHBKFJNZNSGHDS-FGPLHTHASA-N 0.000 description 1
- XYPJXLLXNSAWHZ-SRVKXCTJSA-N Asp-Ser-Tyr Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O XYPJXLLXNSAWHZ-SRVKXCTJSA-N 0.000 description 1
- IQCJOIHDVFJQFV-LKXGYXEUSA-N Asp-Thr-Cys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CC(=O)O)N)O IQCJOIHDVFJQFV-LKXGYXEUSA-N 0.000 description 1
- JJQGZGOEDSSHTE-FOHZUACHSA-N Asp-Thr-Gly Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(O)=O JJQGZGOEDSSHTE-FOHZUACHSA-N 0.000 description 1
- GFYOIYJJMSHLSN-QXEWZRGKSA-N Asp-Val-Arg Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O GFYOIYJJMSHLSN-QXEWZRGKSA-N 0.000 description 1
- 102100021277 Beta-secretase 2 Human genes 0.000 description 1
- 101710150190 Beta-secretase 2 Proteins 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 238000009010 Bradford assay Methods 0.000 description 1
- 101100505161 Caenorhabditis elegans mel-32 gene Proteins 0.000 description 1
- 102100023321 Ceruloplasmin Human genes 0.000 description 1
- 241000699800 Cricetinae Species 0.000 description 1
- MBPKYKSYUAPLMY-DCAQKATOSA-N Cys-Arg-Leu Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(O)=O MBPKYKSYUAPLMY-DCAQKATOSA-N 0.000 description 1
- XZKJEOMFLDVXJG-KATARQTJSA-N Cys-Leu-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)N)O XZKJEOMFLDVXJG-KATARQTJSA-N 0.000 description 1
- GDNWBSFSHJVXKL-GUBZILKMSA-N Cys-Lys-Gln Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(N)=O)C(O)=O GDNWBSFSHJVXKL-GUBZILKMSA-N 0.000 description 1
- YXPNKXFOBHRUBL-BJDJZHNGSA-N Cys-Lys-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CS)N YXPNKXFOBHRUBL-BJDJZHNGSA-N 0.000 description 1
- 241000255925 Diptera Species 0.000 description 1
- JEFZIKRIDLHOIF-BYPYZUCNSA-N Gln-Gly Chemical compound NC(=O)CC[C@H](N)C(=O)NCC(O)=O JEFZIKRIDLHOIF-BYPYZUCNSA-N 0.000 description 1
- BPDVTFBJZNBHEU-HGNGGELXSA-N Glu-Ala-His Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CC1=CN=CN1 BPDVTFBJZNBHEU-HGNGGELXSA-N 0.000 description 1
- SVZIKUHLRKVZIF-GUBZILKMSA-N Glu-Asn-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CCC(=O)O)N SVZIKUHLRKVZIF-GUBZILKMSA-N 0.000 description 1
- AFODTOLGSZQDSL-PEFMBERDSA-N Glu-Asn-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CCC(=O)O)N AFODTOLGSZQDSL-PEFMBERDSA-N 0.000 description 1
- FBEJIDRSQCGFJI-GUBZILKMSA-N Glu-Leu-Ser Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O FBEJIDRSQCGFJI-GUBZILKMSA-N 0.000 description 1
- MFNUFCFRAZPJFW-JYJNAYRXSA-N Glu-Lys-Phe Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 MFNUFCFRAZPJFW-JYJNAYRXSA-N 0.000 description 1
- TWYFJOHWGCCRIR-DCAQKATOSA-N Glu-Pro-Arg Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCNC(N)=N)C(O)=O TWYFJOHWGCCRIR-DCAQKATOSA-N 0.000 description 1
- JLCYOCDGIUZMKQ-JBACZVJFSA-N Glu-Trp-Phe Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)O)NC(=O)[C@H](CC2=CNC3=CC=CC=C32)NC(=O)[C@H](CCC(=O)O)N JLCYOCDGIUZMKQ-JBACZVJFSA-N 0.000 description 1
- DTPOVRRYXPJJAZ-FJXKBIBVSA-N Gly-Arg-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CCCN=C(N)N DTPOVRRYXPJJAZ-FJXKBIBVSA-N 0.000 description 1
- DWUKOTKSTDWGAE-BQBZGAKWSA-N Gly-Asn-Arg Chemical compound NCC(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(O)=O)CCCN=C(N)N DWUKOTKSTDWGAE-BQBZGAKWSA-N 0.000 description 1
- QGZSAHIZRQHCEQ-QWRGUYRKSA-N Gly-Asp-Tyr Chemical compound NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 QGZSAHIZRQHCEQ-QWRGUYRKSA-N 0.000 description 1
- CEXINUGNTZFNRY-BYPYZUCNSA-N Gly-Cys-Gly Chemical compound [NH3+]CC(=O)N[C@@H](CS)C(=O)NCC([O-])=O CEXINUGNTZFNRY-BYPYZUCNSA-N 0.000 description 1
- JSNNHGHYGYMVCK-XVKPBYJWSA-N Gly-Glu-Val Chemical compound [H]NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O JSNNHGHYGYMVCK-XVKPBYJWSA-N 0.000 description 1
- XPJBQTCXPJNIFE-ZETCQYMHSA-N Gly-Gly-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)CNC(=O)CN XPJBQTCXPJNIFE-ZETCQYMHSA-N 0.000 description 1
- FSPVILZGHUJOHS-QWRGUYRKSA-N Gly-His-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CC1=CNC=N1 FSPVILZGHUJOHS-QWRGUYRKSA-N 0.000 description 1
- HHRODZSXDXMUHS-LURJTMIESA-N Gly-Met-Gly Chemical compound CSCC[C@H](NC(=O)C[NH3+])C(=O)NCC([O-])=O HHRODZSXDXMUHS-LURJTMIESA-N 0.000 description 1
- WNZOCXUOGVYYBJ-CDMKHQONSA-N Gly-Phe-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)NC(=O)CN)O WNZOCXUOGVYYBJ-CDMKHQONSA-N 0.000 description 1
- VNNRLUNBJSWZPF-ZKWXMUAHSA-N Gly-Ser-Ile Chemical compound [H]NCC(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O VNNRLUNBJSWZPF-ZKWXMUAHSA-N 0.000 description 1
- POJJAZJHBGXEGM-YUMQZZPRSA-N Gly-Ser-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)CN POJJAZJHBGXEGM-YUMQZZPRSA-N 0.000 description 1
- JSLVAHYTAJJEQH-QWRGUYRKSA-N Gly-Ser-Phe Chemical compound NCC(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 JSLVAHYTAJJEQH-QWRGUYRKSA-N 0.000 description 1
- PYFIQROSWQERAS-LBPRGKRZSA-N Gly-Trp-Gly Chemical compound C1=CC=C2C(C[C@H](NC(=O)CN)C(=O)NCC(O)=O)=CNC2=C1 PYFIQROSWQERAS-LBPRGKRZSA-N 0.000 description 1
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 1
- DZMVESFTHXSSPZ-XVYDVKMFSA-N His-Ala-Ser Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O DZMVESFTHXSSPZ-XVYDVKMFSA-N 0.000 description 1
- UOAVQQRILDGZEN-SRVKXCTJSA-N His-Asp-Leu Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O UOAVQQRILDGZEN-SRVKXCTJSA-N 0.000 description 1
- UPGJWSUYENXOPV-HGNGGELXSA-N His-Gln-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CC1=CN=CN1)N UPGJWSUYENXOPV-HGNGGELXSA-N 0.000 description 1
- VBOFRJNDIOPNDO-YUMQZZPRSA-N His-Gly-Asn Chemical compound C1=C(NC=N1)C[C@@H](C(=O)NCC(=O)N[C@@H](CC(=O)N)C(=O)O)N VBOFRJNDIOPNDO-YUMQZZPRSA-N 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- OVPYIUNCVSOVNF-KQXIARHKSA-N Ile-Gln-Pro Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N1CCC[C@@H]1C(=O)O)N OVPYIUNCVSOVNF-KQXIARHKSA-N 0.000 description 1
- OVPYIUNCVSOVNF-ZPFDUUQYSA-N Ile-Gln-Pro Natural products CC[C@H](C)[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N1CCC[C@H]1C(O)=O OVPYIUNCVSOVNF-ZPFDUUQYSA-N 0.000 description 1
- YHFPHRUWZMEOIX-CYDGBPFRSA-N Ile-Val-Val Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C(C)C)C(=O)O)N YHFPHRUWZMEOIX-CYDGBPFRSA-N 0.000 description 1
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 1
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 1
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 1
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 1
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 1
- SENJXOPIZNYLHU-UHFFFAOYSA-N L-leucyl-L-arginine Natural products CC(C)CC(N)C(=O)NC(C(O)=O)CCCN=C(N)N SENJXOPIZNYLHU-UHFFFAOYSA-N 0.000 description 1
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
- 241000880493 Leptailurus serval Species 0.000 description 1
- CQQGCWPXDHTTNF-GUBZILKMSA-N Leu-Ala-Glu Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCC(O)=O CQQGCWPXDHTTNF-GUBZILKMSA-N 0.000 description 1
- IWTBYNQNAPECCS-AVGNSLFASA-N Leu-Glu-His Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@H](C(O)=O)CC1=CN=CN1 IWTBYNQNAPECCS-AVGNSLFASA-N 0.000 description 1
- QPXBPQUGXHURGP-UWVGGRQHSA-N Leu-Gly-Met Chemical compound CC(C)C[C@@H](C(=O)NCC(=O)N[C@@H](CCSC)C(=O)O)N QPXBPQUGXHURGP-UWVGGRQHSA-N 0.000 description 1
- VGPCJSXPPOQPBK-YUMQZZPRSA-N Leu-Gly-Ser Chemical compound CC(C)C[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O VGPCJSXPPOQPBK-YUMQZZPRSA-N 0.000 description 1
- BKTXKJMNTSMJDQ-AVGNSLFASA-N Leu-His-Gln Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N BKTXKJMNTSMJDQ-AVGNSLFASA-N 0.000 description 1
- LVTJJOJKDCVZGP-QWRGUYRKSA-N Leu-Lys-Gly Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)NCC(O)=O LVTJJOJKDCVZGP-QWRGUYRKSA-N 0.000 description 1
- BGZCJDGBBUUBHA-KKUMJFAQSA-N Leu-Lys-Leu Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(O)=O BGZCJDGBBUUBHA-KKUMJFAQSA-N 0.000 description 1
- BJWKOATWNQJPSK-SRVKXCTJSA-N Leu-Met-Glu Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N BJWKOATWNQJPSK-SRVKXCTJSA-N 0.000 description 1
- INCJJHQRZGQLFC-KBPBESRZSA-N Leu-Phe-Gly Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)NCC(O)=O INCJJHQRZGQLFC-KBPBESRZSA-N 0.000 description 1
- JLYUZRKPDKHUTC-WDSOQIARSA-N Leu-Pro-Trp Chemical compound [H]N[C@@H](CC(C)C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O JLYUZRKPDKHUTC-WDSOQIARSA-N 0.000 description 1
- LJBVRCDPWOJOEK-PPCPHDFISA-N Leu-Thr-Ile Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O LJBVRCDPWOJOEK-PPCPHDFISA-N 0.000 description 1
- ILDSIMPXNFWKLH-KATARQTJSA-N Leu-Thr-Ser Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(O)=O ILDSIMPXNFWKLH-KATARQTJSA-N 0.000 description 1
- AIMGJYMCTAABEN-GVXVVHGQSA-N Leu-Val-Glu Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O AIMGJYMCTAABEN-GVXVVHGQSA-N 0.000 description 1
- QESXLSQLQHHTIX-RHYQMDGZSA-N Leu-Val-Thr Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O QESXLSQLQHHTIX-RHYQMDGZSA-N 0.000 description 1
- NCTDKZKNBDZDOL-GARJFASQSA-N Lys-Asn-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC(=O)N)NC(=O)[C@H](CCCCN)N)C(=O)O NCTDKZKNBDZDOL-GARJFASQSA-N 0.000 description 1
- YVMQJGWLHRWMDF-MNXVOIDGSA-N Lys-Gln-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CCCCN)N YVMQJGWLHRWMDF-MNXVOIDGSA-N 0.000 description 1
- MYZMQWHPDAYKIE-SRVKXCTJSA-N Lys-Leu-Ala Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(O)=O MYZMQWHPDAYKIE-SRVKXCTJSA-N 0.000 description 1
- XYLSGAWRCZECIQ-JYJNAYRXSA-N Lys-Tyr-Glu Chemical compound NCCCC[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](CCC(O)=O)C(O)=O)CC1=CC=C(O)C=C1 XYLSGAWRCZECIQ-JYJNAYRXSA-N 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 101150037717 Mavs gene Proteins 0.000 description 1
- 201000009906 Meningitis Diseases 0.000 description 1
- CEGVMWAVGBRVFS-XGEHTFHBSA-N Met-Cys-Thr Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CS)C(=O)N[C@@H]([C@@H](C)O)C(O)=O CEGVMWAVGBRVFS-XGEHTFHBSA-N 0.000 description 1
- OGAZPKJHHZPYFK-GARJFASQSA-N Met-Glu-Pro Chemical compound CSCC[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N1CCC[C@@H]1C(=O)O)N OGAZPKJHHZPYFK-GARJFASQSA-N 0.000 description 1
- YBAFDPFAUTYYRW-UHFFFAOYSA-N N-L-alpha-glutamyl-L-leucine Natural products CC(C)CC(C(O)=O)NC(=O)C(N)CCC(O)=O YBAFDPFAUTYYRW-UHFFFAOYSA-N 0.000 description 1
- SITLTJHOQZFJGG-UHFFFAOYSA-N N-L-alpha-glutamyl-L-valine Natural products CC(C)C(C(O)=O)NC(=O)C(N)CCC(O)=O SITLTJHOQZFJGG-UHFFFAOYSA-N 0.000 description 1
- XMBSYZWANAQXEV-UHFFFAOYSA-N N-alpha-L-glutamyl-L-phenylalanine Natural products OC(=O)CCC(N)C(=O)NC(C(O)=O)CC1=CC=CC=C1 XMBSYZWANAQXEV-UHFFFAOYSA-N 0.000 description 1
- KZNQNBZMBZJQJO-UHFFFAOYSA-N N-glycyl-L-proline Natural products NCC(=O)N1CCCC1C(O)=O KZNQNBZMBZJQJO-UHFFFAOYSA-N 0.000 description 1
- AJHCSUXXECOXOY-UHFFFAOYSA-N N-glycyl-L-tryptophan Natural products C1=CC=C2C(CC(NC(=O)CN)C(O)=O)=CNC2=C1 AJHCSUXXECOXOY-UHFFFAOYSA-N 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
- 108090001074 Nucleocapsid Proteins Proteins 0.000 description 1
- 201000005702 Pertussis Diseases 0.000 description 1
- UUWCIPUVJJIEEP-SRVKXCTJSA-N Phe-Asn-Cys Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CS)C(=O)O)N UUWCIPUVJJIEEP-SRVKXCTJSA-N 0.000 description 1
- KYYMILWEGJYPQZ-IHRRRGAJSA-N Phe-Glu-Glu Chemical compound OC(=O)CC[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)CC1=CC=CC=C1 KYYMILWEGJYPQZ-IHRRRGAJSA-N 0.000 description 1
- KRYSMKKRRRWOCZ-QEWYBTABSA-N Phe-Ile-Glu Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCC(O)=O)C(O)=O KRYSMKKRRRWOCZ-QEWYBTABSA-N 0.000 description 1
- CDHURCQGUDNBMA-UBHSHLNASA-N Phe-Val-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CC1=CC=CC=C1 CDHURCQGUDNBMA-UBHSHLNASA-N 0.000 description 1
- UVKNEILZSJMKSR-FXQIFTODSA-N Pro-Asn-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H]1CCCN1 UVKNEILZSJMKSR-FXQIFTODSA-N 0.000 description 1
- AUQGUYPHJSMAKI-CYDGBPFRSA-N Pro-Ile-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@@H]1CCCN1 AUQGUYPHJSMAKI-CYDGBPFRSA-N 0.000 description 1
- AJBQTGZIZQXBLT-STQMWFEESA-N Pro-Phe-Gly Chemical compound C([C@@H](C(=O)NCC(=O)O)NC(=O)[C@H]1NCCC1)C1=CC=CC=C1 AJBQTGZIZQXBLT-STQMWFEESA-N 0.000 description 1
- FDMCIBSQRKFSTJ-RHYQMDGZSA-N Pro-Thr-Leu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(O)=O FDMCIBSQRKFSTJ-RHYQMDGZSA-N 0.000 description 1
- GZNYIXWOIUFLGO-ZJDVBMNYSA-N Pro-Thr-Thr Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O GZNYIXWOIUFLGO-ZJDVBMNYSA-N 0.000 description 1
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 1
- BNFVPSRLHHPQKS-WHFBIAKZSA-N Ser-Asp-Gly Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(O)=O BNFVPSRLHHPQKS-WHFBIAKZSA-N 0.000 description 1
- MPPHJZYXDVDGOF-BWBBJGPYSA-N Ser-Cys-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H](CS)NC(=O)[C@@H](N)CO MPPHJZYXDVDGOF-BWBBJGPYSA-N 0.000 description 1
- CRZRTKAVUUGKEQ-ACZMJKKPSA-N Ser-Gln-Ala Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C)C(O)=O CRZRTKAVUUGKEQ-ACZMJKKPSA-N 0.000 description 1
- SQBLRDDJTUJDMV-ACZMJKKPSA-N Ser-Glu-Asn Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O SQBLRDDJTUJDMV-ACZMJKKPSA-N 0.000 description 1
- GZFAWAQTEYDKII-YUMQZZPRSA-N Ser-Gly-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CO GZFAWAQTEYDKII-YUMQZZPRSA-N 0.000 description 1
- ZOPISOXXPQNOCO-SVSWQMSJSA-N Ser-Ile-Thr Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)O)NC(=O)[C@H](CO)N ZOPISOXXPQNOCO-SVSWQMSJSA-N 0.000 description 1
- KCNSGAMPBPYUAI-CIUDSAMLSA-N Ser-Leu-Asn Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O KCNSGAMPBPYUAI-CIUDSAMLSA-N 0.000 description 1
- GZSZPKSBVAOGIE-CIUDSAMLSA-N Ser-Lys-Ala Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(O)=O GZSZPKSBVAOGIE-CIUDSAMLSA-N 0.000 description 1
- NQZFFLBPNDLTPO-DLOVCJGASA-N Ser-Phe-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)NC(=O)[C@H](CO)N NQZFFLBPNDLTPO-DLOVCJGASA-N 0.000 description 1
- UPLYXVPQLJVWMM-KKUMJFAQSA-N Ser-Phe-Leu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(C)C)C(O)=O UPLYXVPQLJVWMM-KKUMJFAQSA-N 0.000 description 1
- PYTKULIABVRXSC-BWBBJGPYSA-N Ser-Ser-Thr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(O)=O PYTKULIABVRXSC-BWBBJGPYSA-N 0.000 description 1
- BDMWLJLPPUCLNV-XGEHTFHBSA-N Ser-Thr-Val Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(O)=O BDMWLJLPPUCLNV-XGEHTFHBSA-N 0.000 description 1
- HAYADTTXNZFUDM-IHRRRGAJSA-N Ser-Tyr-Val Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](C(C)C)C(O)=O HAYADTTXNZFUDM-IHRRRGAJSA-N 0.000 description 1
- PMTWIUBUQRGCSB-FXQIFTODSA-N Ser-Val-Ala Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C)C(O)=O PMTWIUBUQRGCSB-FXQIFTODSA-N 0.000 description 1
- LGIMRDKGABDMBN-DCAQKATOSA-N Ser-Val-Lys Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CO)N LGIMRDKGABDMBN-DCAQKATOSA-N 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- 239000012505 Superdex™ Substances 0.000 description 1
- 108010008038 Synthetic Vaccines Proteins 0.000 description 1
- GKMYGVQDGVYCPC-IUKAMOBKSA-N Thr-Asp-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H]([C@@H](C)O)N GKMYGVQDGVYCPC-IUKAMOBKSA-N 0.000 description 1
- RRRRCRYTLZVCEN-HJGDQZAQSA-N Thr-Leu-Asp Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O RRRRCRYTLZVCEN-HJGDQZAQSA-N 0.000 description 1
- MROIJTGJGIDEEJ-RCWTZXSCSA-N Thr-Pro-Pro Chemical compound C[C@@H](O)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N1[C@H](C(O)=O)CCC1 MROIJTGJGIDEEJ-RCWTZXSCSA-N 0.000 description 1
- WPSKTVVMQCXPRO-BWBBJGPYSA-N Thr-Ser-Ser Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O WPSKTVVMQCXPRO-BWBBJGPYSA-N 0.000 description 1
- ZMYCLHFLHRVOEA-HEIBUPTGSA-N Thr-Thr-Ser Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(O)=O ZMYCLHFLHRVOEA-HEIBUPTGSA-N 0.000 description 1
- COYHRQWNJDJCNA-NUJDXYNKSA-N Thr-Thr-Thr Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O COYHRQWNJDJCNA-NUJDXYNKSA-N 0.000 description 1
- GRIUMVXCJDKVPI-IZPVPAKOSA-N Thr-Thr-Tyr Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O GRIUMVXCJDKVPI-IZPVPAKOSA-N 0.000 description 1
- KZTLZZQTJMCGIP-ZJDVBMNYSA-N Thr-Val-Thr Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O KZTLZZQTJMCGIP-ZJDVBMNYSA-N 0.000 description 1
- FNOQJVHFVLVMOS-AAEUAGOBSA-N Trp-Gly-Asn Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)NCC(=O)N[C@@H](CC(=O)N)C(=O)O)N FNOQJVHFVLVMOS-AAEUAGOBSA-N 0.000 description 1
- HABYQJRYDKEVOI-IHPCNDPISA-N Trp-His-Lys Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CC3=CN=CN3)C(=O)N[C@@H](CCCCN)C(=O)O)N HABYQJRYDKEVOI-IHPCNDPISA-N 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- BODHJXJNRVRKFA-BZSNNMDCSA-N Tyr-Cys-Tyr Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CS)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O BODHJXJNRVRKFA-BZSNNMDCSA-N 0.000 description 1
- ZSXJENBJGRHKIG-UWVGGRQHSA-N Tyr-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 ZSXJENBJGRHKIG-UWVGGRQHSA-N 0.000 description 1
- VYQQQIRHIFALGE-UWJYBYFXSA-N Tyr-Ser-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 VYQQQIRHIFALGE-UWJYBYFXSA-N 0.000 description 1
- SOAUMCDLIUGXJJ-SRVKXCTJSA-N Tyr-Ser-Asn Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(O)=O SOAUMCDLIUGXJJ-SRVKXCTJSA-N 0.000 description 1
- ZPFLBLFITJCBTP-QWRGUYRKSA-N Tyr-Ser-Gly Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CO)C(=O)NCC(O)=O ZPFLBLFITJCBTP-QWRGUYRKSA-N 0.000 description 1
- -1 Val Natural products 0.000 description 1
- VMRFIKXKOFNMHW-GUBZILKMSA-N Val-Arg-Ser Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CO)C(=O)O)N VMRFIKXKOFNMHW-GUBZILKMSA-N 0.000 description 1
- IDKGBVZGNTYYCC-QXEWZRGKSA-N Val-Asn-Pro Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)N1CCC[C@H]1C(O)=O IDKGBVZGNTYYCC-QXEWZRGKSA-N 0.000 description 1
- TZVUSFMQWPWHON-NHCYSSNCSA-N Val-Asp-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](C(C)C)N TZVUSFMQWPWHON-NHCYSSNCSA-N 0.000 description 1
- CELJCNRXKZPTCX-XPUUQOCRSA-N Val-Gly-Ala Chemical compound CC(C)[C@H](N)C(=O)NCC(=O)N[C@@H](C)C(O)=O CELJCNRXKZPTCX-XPUUQOCRSA-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
- PMDOQZFYGWZSTK-LSJOCFKGSA-N Val-Gly-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)C(C)C PMDOQZFYGWZSTK-LSJOCFKGSA-N 0.000 description 1
- OPGWZDIYEYJVRX-AVGNSLFASA-N Val-His-Arg Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)N OPGWZDIYEYJVRX-AVGNSLFASA-N 0.000 description 1
- PTFPUAXGIKTVNN-ONGXEEELSA-N Val-His-Gly Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)NCC(=O)O)N PTFPUAXGIKTVNN-ONGXEEELSA-N 0.000 description 1
- QPPZEDOTPZOSEC-RCWTZXSCSA-N Val-Met-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CCSC)NC(=O)[C@H](C(C)C)N)O QPPZEDOTPZOSEC-RCWTZXSCSA-N 0.000 description 1
- DLRZGNXCXUGIDG-KKHAAJSZSA-N Val-Thr-Asp Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)O)NC(=O)[C@H](C(C)C)N)O DLRZGNXCXUGIDG-KKHAAJSZSA-N 0.000 description 1
- NLNCNKIVJPEFBC-DLOVCJGASA-N Val-Val-Glu Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CCC(O)=O NLNCNKIVJPEFBC-DLOVCJGASA-N 0.000 description 1
- 108700005077 Viral Genes Proteins 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 235000004279 alanine Nutrition 0.000 description 1
- 108010024078 alanyl-glycyl-serine Proteins 0.000 description 1
- 108010086434 alanyl-seryl-glycine Proteins 0.000 description 1
- 108010044940 alanylglutamine Proteins 0.000 description 1
- 108010087924 alanylproline Proteins 0.000 description 1
- 230000007815 allergy Effects 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 108010013835 arginine glutamate Proteins 0.000 description 1
- 125000000637 arginyl group Chemical group N[C@@H](CCCNC(N)=N)C(=O)* 0.000 description 1
- 108010010430 asparagine-proline-alanine Proteins 0.000 description 1
- 229940009098 aspartate Drugs 0.000 description 1
- 108010069205 aspartyl-phenylalanine Proteins 0.000 description 1
- 108010092854 aspartyllysine Proteins 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000002238 attenuated effect Effects 0.000 description 1
- 229940031567 attenuated vaccine Drugs 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000000740 bleeding effect Effects 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 210000001728 clone cell Anatomy 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 108091036078 conserved sequence Proteins 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 108010016616 cysteinylglycine Proteins 0.000 description 1
- 108010060199 cysteinylproline Proteins 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000002405 diagnostic procedure Methods 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- 108010054812 diprotin A Proteins 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 229960003239 encephalitis vaccine Drugs 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 239000006167 equilibration buffer Substances 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000001125 extrusion Methods 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- 108010008237 glutamyl-valyl-glycine Proteins 0.000 description 1
- 108010079547 glutamylmethionine Proteins 0.000 description 1
- 108010019407 glycyl-arginyl-glycyl-aspartic acid Proteins 0.000 description 1
- 108010074027 glycyl-seryl-phenylalanine Proteins 0.000 description 1
- 108010089804 glycyl-threonine Proteins 0.000 description 1
- 108010015792 glycyllysine Proteins 0.000 description 1
- 108010084389 glycyltryptophan Proteins 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000035931 haemagglutination Effects 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 108010036413 histidylglycine Proteins 0.000 description 1
- 108010028295 histidylhistidine Proteins 0.000 description 1
- 108010092114 histidylphenylalanine Proteins 0.000 description 1
- 230000002519 immonomodulatory effect Effects 0.000 description 1
- 230000036737 immune function Effects 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 229960001438 immunostimulant agent Drugs 0.000 description 1
- 239000003022 immunostimulating agent Substances 0.000 description 1
- 230000003308 immunostimulating effect Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 238000010255 intramuscular injection Methods 0.000 description 1
- 239000007927 intramuscular injection Substances 0.000 description 1
- 210000003292 kidney cell Anatomy 0.000 description 1
- 108010083708 leucyl-aspartyl-valine Proteins 0.000 description 1
- 108010034529 leucyl-lysine Proteins 0.000 description 1
- 108010000761 leucylarginine Proteins 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 229960001453 live attenuated japanese encephalitis Drugs 0.000 description 1
- 210000002751 lymph Anatomy 0.000 description 1
- 108010017391 lysylvaline Proteins 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 108010005942 methionylglycine Proteins 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 239000013610 patient sample Substances 0.000 description 1
- 108010074082 phenylalanyl-alanyl-lysine Proteins 0.000 description 1
- 108010064486 phenylalanyl-leucyl-valine Proteins 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 108010077112 prolyl-proline Proteins 0.000 description 1
- 108010070643 prolylglutamic acid Proteins 0.000 description 1
- 229940124551 recombinant vaccine Drugs 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 239000012898 sample dilution Substances 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000000405 serological effect Effects 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 108020001568 subdomains Proteins 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 108010072986 threonyl-seryl-lysine Proteins 0.000 description 1
- 238000002255 vaccination Methods 0.000 description 1
- 229940125575 vaccine candidate Drugs 0.000 description 1
- 210000003501 vero cell Anatomy 0.000 description 1
- 210000000605 viral structure Anatomy 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- 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
- A61K39/12—Viral antigens
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1081—Togaviridae, e.g. flavivirus, rubella virus, hog cholera virus
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- 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
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/24011—Flaviviridae
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/18—Togaviridae; Flaviviridae
- G01N2333/183—Flaviviridae, e.g. pestivirus, mucosal disease virus, bovine viral diarrhoea virus, classical swine fever virus (hog cholera virus) or border disease virus
- G01N2333/185—Flaviviruses or Group B arboviruses, e.g. yellow fever virus, japanese encephalitis, tick-borne encephalitis, dengue
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Virology (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Pharmacology & Pharmacy (AREA)
- Hematology (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Urology & Nephrology (AREA)
- Gastroenterology & Hepatology (AREA)
- Biomedical Technology (AREA)
- Mycology (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Food Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Peptides Or Proteins (AREA)
Abstract
Description
본 발명은 일본 뇌염 바이러스 피막 단백질의 돌출 도메인을 포함하는 단백질, 이의 동형이량체, 및 이의 용도에 관한 것이다.The present invention relates to a protein comprising a protruding domain of a Japanese encephalitis virus coat protein, a homodimer thereof, and uses thereof.
일본 뇌염 바이러스(japanese encephalitis virus, JEV)는 플라비비리데과(Flaviviridae), 플라비바이러스속(Flavivirus)에 속하는 바이러스로, 40 nm 내지 50 nm의 직경을 가진 정이십면체의 바이러스이다. 바이러스 표면은 막 단백질(membrane protein, M), 피막 단백질(envelope protein, E), 및 지질로 구성된 피막으로 구성되어 있다. 상기 바이러스 유전자는 양성 단일 가닥 RNA를 가지고 있으며 크기는 약 11 kb 이다.Japanese encephalitis virus (japanese encephalitis virus, JEV) is a Flaviviridae degwa (Flaviviridae), flaviviruses in positive virus of twenty-sided with a diameter of a virus, 40 nm to 50 nm that belongs to (Flavivirus). The virus surface consists of a membrane protein consisting of membrane protein (M), envelope protein (E), and lipid. The viral gene has a positive single stranded RNA and is about 11 kb in size.
일반적으로 JEV는 감염 시 뇌염(encephalitis), 무균성 수막염(aseptic meningtis), 열성 두통(febrile headache)의 증상을 보인다. 병의 증세는 나이가 증가함에 따라 증가하며 60세 이상의 고령자에서는 높은 비율로 뇌염을 일으킨다. JEV는 한국, 일본, 중국, 대만, 동남아시아, 인도, 러시아를 포함하는 아시아 전역에 널리 퍼져있으며 최근에는 구미지역에서도 출현이 보고되고 있다. 그러나, 현재까지 뚜렷한 치료방법은 알려진 것이 없다.In general, JEV presents with symptoms of encephalitis, aseptic meningitis, febrile headache. Symptoms of the disease increase with age and elderly people over 60 years old cause a high rate of encephalitis. JEV has spread throughout Asia, including Korea, Japan, China, Taiwan, Southeast Asia, India and Russia. However, until now, no obvious treatment has been known.
일본 뇌염 바이러스 백신은 쥐 뇌조직 유래 백신 및 햄스터 신장세포 유래 백신과 같은 2 종류의 불활성화 사백신 및 약독화 생백신과 같이 총 3 종류의 백신이 있다. 한편, 우리나라에서는 Nakayama주를 이용한 불활성화 쥐 뇌조직 유래 사백신과 약독화 백신이 사용되고 있다(Min Soo Park, et al., 1999). 현재, 여러 가지 새로운 일본 뇌염 백신이 개발되고 있다. 그 중에 대표적인 백신은 유전자 재조합 백신과 베로 세포(Vero cell) 유래 불활성화 사백신이다. 불활성화 사백신의 접종은 기본 3회 접종 후 각 국가마다 다른 추가접종 방식을 택하고 있다. 불활화 백신은 마우스 내에서 유래 되었기 때문에 드물게 접종자에게 쇼크를 일으킬 수 있고, 면역력이 장기간 유지되지 않기 때문에 2년마다 재접종하여야 하며, 마우스를 사용하기 때문에 경제성의 문제가 있다.The Japanese encephalitis virus vaccine has three kinds of vaccines, such as a rat brain tissue-derived vaccine and a hamster kidney cell-derived vaccine, such as two inactivated vaccines and an attenuated live vaccine. On the other hand, in Korea, inactivated rat brain tissue-derived vaccine using Nakayama strain and attenuated vaccine have been used (Min Soo Park, et al., 1999). Currently, several new Japanese encephalitis vaccines are being developed. Among them, representative vaccines are recombinant vaccines and inactivated vaccines derived from Vero cells. Inoculation of inactivated vaccine has been made after three doses of basic doses. Since the inactivated vaccine is derived from the mouse, it rarely causes shock to the inoculator. Since the immunity is not maintained for a long time, it is required to be re-vaccinated every 2 years.
한편, 일본 뇌염 진단의 목적은 환자의 감염 진단 및 발생 감시로 구분할 수 있다. 환자의 일본 뇌염 바이러스 감염 진단을 위해, 환자 검체에서 바이러스 분리, 혈중 항체가 측정 및 유전자 검출 방법 등을 수행할 수 있다. 일본 뇌염 발생을 감시하기 위해서는, 증폭 숙주인 돼지에서 일본 뇌염 특이항체 측정 및 매개체인 모기에서 바이러스 분리 시험 등을 수행한다. 바이러스에 대한 항체검사 중, 중화항체역가측정시험법은 판정까지의 시간이 많이 걸린다는 단점이 있어 일반적인 진단법으로 사용하기는 어렵다. 또한, 혈구응집억제시험법은 신속하게 일본 뇌염 감염 여부를 확인할 수 있다는 장점이 있지만 시약 제조가 복잡하고 검사자에 따라 결과에 차이가 많이 나는 단점이 있다. 따라서, 일본 뇌염 바이러스 감염 진단을 위한 시험법의 개선이 요구되고 있는 실정이다.On the other hand, the purpose of the diagnosis of Japanese encephalitis can be classified into diagnosis of infection and observation of occurrence of the patient. In order to diagnose a Japanese encephalitis virus infection of a patient, virus isolation, blood antibody measurement and gene detection method can be performed in a patient sample. In order to monitor the development of Japanese encephalitis, a Japanese encephalitis-specific antibody is measured in a pig, which is an amplification host, and a virus isolation test is performed in a mosquito, a mediator. During antibody testing for viruses, the neutralization antibody titers test method has a disadvantage that it takes much time until the determination, and it is difficult to use it as a general diagnostic method. In addition, the hemagglutination inhibition test method has a merit that it can quickly confirm the infection with Japanese encephalitis, but it has a disadvantage that the manufacture of the reagent is complicated and the results are different depending on the examiner. Therefore, there is a need to improve the test method for the diagnosis of Japanese encephalitis virus infection.
이에, 본 발명자들은 일본 뇌염 바이러스 감염에 따른 혈청학적 진단을 위해 일본 뇌염 바이러스의 항원 단백질인 피막 단백질을 구조적 안정화시킨 형태로 곤충세포에 발현 및 정제를 하였다. 이를 ELISA 방법을 통하여 감염 동물의 혈청에서 높은 감도로 항체가를 측정할 수 있었다. 또한, 상기 정제된 재조합 피막 단백질을 실험동물에 면역 후 생성되는 항체가 일본 뇌염 바이러스에 대한 중화능력을 확인함으로써, 본 발명을 완성하였다.Thus, the present inventors have expressed and purified the insect cells in a structurally stabilized form of the coat protein, which is the antigen protein of the Japanese encephalitis virus, for the serological diagnosis of the Japanese encephalitis virus infection. The ELISA method was able to measure antibody titer in sera of infected animals with high sensitivity. In addition, the present invention was completed by confirming neutralizing ability of the antibody produced after immunization of the purified recombinant coat protein into an experimental animal with Japanese encephalitis virus.
본 발명의 목적은 일본 뇌염 바이러스 피막 단백질의 돌출 도메인을 구조적으로 안정화시킨 동형이량체 단백질 항원을 제공하는 것이다.It is an object of the present invention to provide a homodimeric protein antigen which structurally stabilizes the protruding domain of a Japanese encephalitis virus coat protein.
본 발명의 다른 목적은 상기 항원에 대한 항체를 포함하는 일본 뇌염의 진단, 예방 또는 치료용 약학적 조성물을 제공하는 것이다.Another object of the present invention is to provide a pharmaceutical composition for diagnosing, preventing or treating Japanese encephalitis which comprises an antibody against said antigen.
상기 목적을 달성하기 위하여, 본 발명은 일본 뇌염 바이러스 피막 단백질 돌출 도메인 두 개가 결합된 동형이량체 단백질을 제공한다.In order to achieve the above object, the present invention provides a homodimer protein having two Japanese encephalitis virus coat protein prominent domains bound thereto.
또한, 상기 동형이량체 단백질을 유효성분으로 포함하는 일본 뇌염 바이러스 감염에 의한 질환 예방 또는 치료용 백신을 제공한다.Also provided is a vaccine for the prevention or treatment of diseases caused by Japanese encephalitis virus infection comprising the homodimeric protein as an effective ingredient.
또한, 상기 동형이량체 단백질에 특이적으로 결합하는 항체 또는 이의 단편을 제공한다.Also provided are antibodies or fragments thereof that specifically bind to said homodimeric proteins.
또한, 상기 동형이량체 단백질을 포함하는 일본 뇌염 바이러스 감염 진단용 키트을 제공한다.Also provided is a kit for the diagnosis of a Japanese encephalitis virus infection comprising the homodimeric protein.
또한, 상기 동형이량체 단백질에 특이적으로 결합하는 항체를 포함하는 일본 뇌염 바이러스 감염 진단용 키트를 제공한다.Also provided is a kit for diagnosing Japanese encephalitis virus infection comprising an antibody that specifically binds to the homodimeric protein.
또한, 상기 일본 뇌염 바이러스 피막 단백질 돌출 도메인 단량체 단백질을 코딩하는 뉴클레오티드를 포함하는 발현 벡터를 제공한다.Also provided is an expression vector comprising a nucleotide encoding the Japanese encephalitis virus coat protein protruding domain monomer protein.
또한, 상기 발현 벡터로 형질감염된 숙주세포를 제공한다.Also provided is a host cell transfected with the expression vector.
또한, 상기 형질감염된 숙주세포로부터 일본 뇌염 바이러스 피막 단백질 돌출 도메인 단량체 단백질을 수득하는 단계를 포함하는 일본 뇌염 바이러스 피막 단백질 돌출 도메인 단량체 단백질의 제조방법을 제공한다.Also provided is a method for producing a Japanese encephalitis virus coat protein protruding domain monomer protein, comprising the step of obtaining a Japanese encephalitis virus coat protein protruding domain monomer protein from the transfected host cell.
또한, (1) 상기 동형이량체 단백질을 항원으로서 실험동물에 주입하여 면역반응을 유도하는 단계; 및 (2) 상기 실험동물로부터 항체를 회수하는 단계를 포함하는 일본 뇌염 바이러스 항체 제조 방법을 제공한다.(1) inducing an immune response by injecting the homodimer protein as an antigen into an experimental animal; And (2) recovering the antibody from the laboratory animal.
본 발명은 곤충세포 발현 시스템을 이용하여 본래 바이러스의 표면에 존재하는 것과 같이 구조적으로 안정한 돌출 도메인의 동형이량체 단백질 항원을 제공한다. 또한, 상기 항원은 개체에 주사할 경우 개체 내 항체 생성능이 우수하여 기존의 일본 뇌염 사백신보다 안정한 백신으로 이용될 수 있으며, 일본 뇌염의 진단에 이용될 수 있다.The present invention utilizes an insect cell expression system to provide a structurally stable protruding domain homodimeric protein antigen such as is present on the surface of the native virus. In addition, the antigen can be used as a vaccine more stable than the conventional Japanese encephalitis virus vaccine, and can be used for the diagnosis of Japanese encephalitis when injected into an individual.
도 1은 단백질 서열분석 비교 프로그램(Clustal W & ESPript 3.0)을 이용하여 플라비 바이러스의 피막 단백질 아미노산 서열을 비교 분석하여 나타낸 것이다.
도 2는 단백질 서열분석 비교 프로그램(Clustal W & ESPript 3.0)을 이용하여 일본 뇌염 바이러스의 피막 단백질 아미노산 서열을 비교 분석하여 나타낸 것이다. 이때, 일본 뇌염바이러스 피막 단백질 돌출 도메인은 1번부터 401번까지이다.
도 3은 일본 뇌염 바이러스 피막 단백질 돌출 도메인의 발현을 웨스턴 블롯을 이용하여 확인한 것이다.
도 4a는 Ni-NTA 친화성 크로마토그래피 기법을 이용하여 일본 뇌염 바이러스 피막 단백질 돌출 도메인을 정제한 후, 순도를 측정한 것이다.
도 4b는 Ni-NTA 친화성 크로마토그래피 기법을 이용하여 일본 뇌염 바이러스 피막 단백질 돌출 도메인을 정제한 후, 돌출 도메인 단백질 발현을 확인한 것이다.
도 4c는 젤 필트레이션 크로마토그래피 기법을 이용하여 일본 뇌염 바이러스 피막 단백질 돌출 도메인을 정제한 후, 순도를 측정한 것이다.
도 4d는 젤 필트레이션 크로마토그래피 기법을 이용하여 일본 뇌염 바이러스 피막 단백질 돌출 도메인을 정제한 후, 돌출 도메인 단백질 발현을 확인한 것이다.
도 4e는 정제된 일본 뇌염 바이러스 피막 단백질 돌출 도메인의 발현을 SDS-PAGE 상에서 확인한 것이다.
도 5는 일본 뇌염 바이러스 피막 단백질 돌출 도메인 항원을 면역한 4마리의 마우스로부터 최종 혈청을 분리한 후, 상기 항원에 의한 면역 항체 형성 정도를 나타낸 것이다.
도 6는 일본 뇌염 바이러스 피막 단백질 돌출 도메인 항원을 2번(JEVx2) 및 3번(JEVx3) 투여한 각 마우스의 혈액 속 항체량을 측정한 것이다.
도 7은 곤충세포 유래 일본 뇌염 바이러스 피막 단백질 돌출 도메인 항원(JEV-E)과 대장균 발현 시스템 유래 항원(LysRS-JEV-E)의 특이도 및 민감도를 측정한 것이다.
도 8a는 면역증강제와 백신의 접종 방법에 따른 항체 생성 정도를 측정한 것이다.
도 8b는 면역증강제와 백신의 접종 방법에 따라 생성된 항체량을 마우스 표준 IgG값을 기준으로 정량한 값이다.FIG. 1 shows the amino acid sequences of the Flaviviruses by comparing the amino acid sequences of the Flaviviruses using a protein sequence analysis program (Clustal W & ESPript 3.0).
FIG. 2 shows the amino acid sequence of the envelope protein of Japanese encephalitis virus using comparative protein sequence analysis program (Clustal W & ESPript 3.0). At this time, the Japanese encephalitis virus coat protein protruding domains are from 1 to 401.
Fig. 3 shows the expression of the Japanese encephalitis virus coat protein overexpression domain using Western blotting.
FIG. 4A shows the purity of the Japanese encephalitis virus coat protein protruding domains purified using a Ni-NTA affinity chromatography technique.
FIG. 4B shows the results of confirming the expression of the protruding domain protein after purifying the protruding domain of Japanese encephalitis virus coat protein using the Ni-NTA affinity chromatography technique.
FIG. 4C shows the purity of the Japanese encephalitis virus coat protein protruding domain purified by gel filtration chromatography.
FIG. 4D shows the appearance of the protruding domain protein after purification of the protruding domain of the Japanese encephalitis virus coat protein using a gel filtration chromatography technique.
4E shows the expression of purified Japanese encephalitis virus coat protein overexpression domain on SDS-PAGE.
5 shows the degree of immune antibody formation by the antigen after separating the final serum from 4 mice immunized with Japanese encephalitis virus coat protein prominent domain antigen.
FIG. 6 is a graph showing the amount of antibodies in the blood of mice injected with the Japanese encephalitis virus coat protein prominent domain antigen 2 (JEVx2) and 3 (JEVx3).
FIG. 7 shows the specificity and sensitivity of the Japanese encephalitis virus coat protein protruding domain antigen (JEV-E) derived from insect cells and the antigen derived from Escherichia coli expression system (LysRS-JEV-E).
FIG. 8A shows the degree of antibody production according to the inoculation method of the immunopotentiator and the vaccine.
FIG. 8B is a value obtained by quantifying the amount of antibody produced according to the inoculation method of the immunopotentiator and the vaccine based on the mouse standard IgG value.
본 발명은 일 측면으로, 일본 뇌염 바이러스 피막 단백질 돌출 도메인 두 개가 결합된 동형이량체 단백질을 제공한다.In one aspect, the present invention provides a homodimeric protein in which two Japanese encephalitis virus coat protein projecting domains are bound.
본 명세서에서 사용된 용어, "일본 뇌염 바이러스"는 플라비비리데과 플라비바이러스 속에 속하는 바이러스이다. 상기 바이러스는 genomic RNA와 캡시드 단백질의 복합체로 구성된 뉴클레오캡시드 및 이를 둘러싼 피막 단백질로 구성되어 있다. 상기 피막 단백질은 바이러스의 혈청형을 결정하는 요소이다.As used herein, the term " Japanese encephalitis virus " is a virus belonging to the genus Flaviviridae and Flaviviridae. The virus is composed of a nucleocapsid composed of a complex of a genomic RNA and a capsid protein and a surrounding membrane protein. The coat protein is a factor that determines the serotype of the virus.
또한, 본 명세서에서 사용된 용어, "일본 뇌염 바이러스 피막 단백질"은 일본 뇌염 바이러스 감염의 중요한 역할을 하며, 상기 바이러스의 활성을 저해하는 백신이나 항체의 타겟이다. 따라서, 상기 피막 단백질은 일본 뇌염의 예방 또는 치료에 유용하게 이용될 수 있다.As used herein, the term " Japanese encephalitis virus coat protein " plays an important role in Japanese encephalitis virus infection and is a target of a vaccine or an antibody that inhibits the activity of the virus. Therefore, the coating protein can be usefully used for the prevention or treatment of Japanese encephalitis.
상기 돌출 도메인은 서열번호 3의 아미노산 서열을 갖는 폴리펩티드 또는 이의 단편일 수 있고, 상기 폴리펩티드는 서열번호 2의 염기서열에 의해 코딩될 수 있다.The protruding domain may be a polypeptide having the amino acid sequence of SEQ ID NO: 3 or a fragment thereof, and the polypeptide may be encoded by the nucleotide sequence of SEQ ID NO: 2.
상기 돌출 도메인은 서브도메인 1(FNCLGMGNRD, (서열번호 4)), 서브도메인 2(VLEGDSCLT, (서열번호 5)), 서브도메인 3(HASVTDIS, (서열번호 6)), 서브도메인 4(GWGNGCGL, (서열번호 7)), 서브도메인 5(TTTSENHGN, (서열번호 8)), 서브도메인 6(AHATKQSVVA, (서열번호 9)), 서브도메인 7(QEGGLHQALAGAIVVEYS, (서열번호 10)), 및 서브도메인 8(DTGHGTV, (서열번호 11))의 아미노산 서열을 포함할 수 있다.The protruding domains include the subdomains 1 (FNCLGMGNRD, (SEQ ID NO: 4), VLEGDSCLT (SEQ ID NO: 5), HASVTDIS (SEQ ID NO: 6), GWGNGCGL (SEQ ID NO: 7), Subdomain 5 (TTTSENHGN, SEQ ID NO: 8), Subdomain 6 (AHATKQSVVA, SEQ ID NO: 9), Subdomain 7 (QEGGLHQALAGAIVVEYS, DTGHGTV, (SEQ ID NO: 11)).
구체적으로, 일본 뇌염 바이러스 피막 단백질 돌출 도메인을 나타내는 서열번호 3의 아미노산 서열에 있어서, 서브도메인 1은 3번 내지 12번에 위치할 수 있고; 서브도메인 2는 26번 내지 34번에 위치할 수 있으며; 서브도메인 3은 64번 내지 71번에 위치할 수 있고; 서브도메인 4는 102번 내지 109번에 위치할 수 있으며; 서브도메인 5는 148번 내지 156번에 위치할 수 있고; 서브도메인 6은 247번 내지 256번에 위치할 수 있으며; 서브도메인 7은 260번 내지 277번에 위치할 수 있고; 서브도메인 8은 318번 내지 324번에 위치할 수 있다.Specifically, in the amino acid sequence of SEQ ID NO: 3 representing the Japanese encephalitis virus coat protein protruding domain,
상기 서브도메인 1 내지 서브도메인 8은 링커를 통해 연결될 수 있다.The
이때, 서브도메인 1과 서브도메인 2를 연결하는 링커(L1)는 1개 내지 30개, 2개 내지 25개, 3개 내지 20개, 또는 5개 내지 15개의 아미노산을 포함할 수 있으며, 바람직하게는 13개의 아미노산으로 구성된 펩티드일 수 있다. 본 발명의 일 실시예에서는, 상기 L1이 서열번호 3의 13번 내지 25번에 위치하였다.At this time, the linker (L1) connecting the
상기 서브도메인 2와 서브도메인 3을 연결하는 링커(L2)는 1개 내지 50개, 2개 내지 45개, 3개 내지 40개, 5개 내지 35개, 또는 10개 내지 30개의 아미노산을 포함할 수 있으며, 바람직하게는 29개의 아미노산으로 구성된 펩티드일 수 있다. 본 발명의 일 실시예에서는, 상기 L2가 서열번호 3의 35번 내지 63번에 위치하였다.Linker
상기 서브도메인 3과 서브도메인 4를 연결하는 링커(L3)는 1개 내지 50개, 2개 내지 45개, 3개 내지 40개, 5개 내지 35개, 또는 10개 내지 30개의 아미노산을 포함할 수 있으며, 바람직하게는 30개의 아미노산으로 구성된 펩티드일 수 있다. 본 발명의 일 실시예에서는, 상기 L3가 서열번호 3의 72번 내지 101번에 위치하였다.The linker (L3) connecting the
상기 서브도메인 4와 서브도메인 5를 연결하는 링커(L4)는 1개 내지 60개, 2개 내지 55개, 3개 내지 50개, 5개 내지 45개, 또는 10개 내지 40개의 아미노산을 포함할 수 있으며, 바람직하게는 37의 아미노산으로 구성된 펩티드일 수 있다. 본 발명의 일 실시예에서는, 상기 L4가 서열번호 3의 110번 내지 147번에 위치하였다.The linker (L4) connecting the
상기 서브도메인 5와 서브도메인 6을 연결하는 링커(L5)는 1개 내지 120개, 20개 내지 110개, 40개 내지 100개, 또는 60개 내지 90개의 아미노산을 포함할 수 있으며, 바람직하게는 90개의 아미노산으로 구성된 펩티드일 수 있다. 본 발명의 일 실시예에서는, 상기 L5가 서열번호 3의 157번 내지 246번에 위치하였다.The linker L5 connecting the
상기 서브도메인 6과 서브도메인 7을 연결하는 링커(L6)는 1개 내지 10개, 2개 내지 8개, 또는 3개 내지 5개의 아미노산을 포함할 수 있으며, 바람직하게는 3개의 아미노산으로 구성된 펩티드일 수 있다. 본 발명의 일 실시예에서는, 상기 L6가 서열번호 3의 257번 내지 259번에 위치하였다.The linker L6 connecting the
상기 서브도메인 7과 서브도메인 8을 연결하는 링커(L7)는 1개 내지 60개, 5개 내지 55개, 10개 내지 50개, 또는 20개 내지 40개의 아미노산을 포함할 수 있으며, 바람직하게는 40개의 아미노산으로 구성된 펩티드일 수 있다. 본 발명의 일 실시예에서는, 상기 L7이 서열번호 3의 278번 내지 317번에 위치하였다.The linker L7 connecting the
상기 링커를 구성하는 아미노산은 아르기닌(Arg), 히스티딘(His), 리신(Lys), 아스파르트산(Asp), 글루탐산(Glu), 세린(Ser), 트레오닌(Thr), 아스파라긴(Asn), 글루타민(Gln), 티로신(Tyr), 알라닌(Ala), 루신(Leu), 아이소루신(Ile), 발린(Val), 페닐알라닌(Phe), 메티오닌(Met), 트립토판(Trp), 글리신(Gly), 프롤린(Pro), 및 시스테인(Cys)으로 구성된 군에서 선택되는 어느 하나 이상일 수 있다.The amino acid of the linker is selected from the group consisting of arginine, histidine, lysine, aspartate, glutamic acid, serine, Gln, Tyr, Alanine, Leu, Ile, Val, Phe, Methionine, Tryptophan, Gly, Proline (Pro), and cysteine (Cys).
본 발명은 다른 측면으로, 상기 일본 뇌염 바이러스 피막 단백질 돌출 도메인을 포함하는 단백질 두 개가 결합된 일본 뇌염 바이러스 피막 단백질 돌출 도메인의 동형이량체 단백질을 제공한다. 상기 결합은 상기 돌출 도메인에 존재하는 서열번호 4 내지 서열번호 10의 부위에서 이루어질 수 있다.In another aspect, the present invention provides a homodimer protein of the Japanese encephalitis virus coat protein prominent domain, wherein two proteins comprising the Japanese encephalitis virus coat protein prominent domain are combined. The linkage may be made at the sites of SEQ ID NOS: 4 to 10 present in the protruding domain.
또한, 본 발명은 상기 동형이량체 단백질을 유효성분으로 포함하는 일본 뇌염 바이러스 감염에 의한 질환 예방 또는 치료용 백신을 제공한다.The present invention also provides a vaccine for the prevention or treatment of diseases caused by Japanese encephalitis virus infection comprising the homodimeric protein as an active ingredient.
상기 일본 뇌염 바이러스 감염에 의한 질환은 뇌염, 무균성수막염, 열성두통 및 이들의 조합으로 구성된 군으로부터 선택되는 것일 수 있다.The disease caused by the Japanese encephalitis virus infection may be selected from the group consisting of encephalitis, aphthous meningitis, febrile headache, and combinations thereof.
상기 약학 조성물은 약학적으로 허용가능한 담체를 추가적으로 포함할 수 있다. 상기 담체는 본 발명의 약학 조성물 총 중량을 기준으로 약 1 중량% 내지 약 99.99 중량%, 5 중량% 내지 90 중량%, 10 중량% 내지 85 중량%, 20 중량% 내지 60중량%, 바람직하게는 약 90 중량% 내지 약 99.99 중량%로 포함될 수 있다.The pharmaceutical composition may additionally comprise a pharmaceutically acceptable carrier. The carrier may be present in an amount of from about 1% to about 99.99%, from 5% to 90%, from 10% to 85%, from 20% to 60% by weight, From about 90% to about 99.99% by weight.
또한, 본 발명은 상기 백신을 개체에 투여하는 단계를 포함하는 일본 뇌염 바이러스 감염에 의한 질환 예방 또는 치료 방법을 제공한다.The present invention also provides a method of preventing or treating a disease caused by Japanese encephalitis virus infection comprising the step of administering the vaccine to an individual.
또한, 본 발명은 상기 동형이량체 단백질에 특이적으로 결합하는 항체 또는 이의 단편을 제공한다.The present invention also provides an antibody or fragment thereof that specifically binds to the homodimeric protein.
상기 용어 "항체"는 면역계 내에서 항원의 자극에 의하여 만들어지는 성분으로서 특정한 항원과 특이적으로 결합하여 림프와 혈액을 떠돌며 항원-항체반응을 일으키는 단백질이다. 항원-항체 반응은 각 항원에 대하여 높은 특이성을 갖는다. 이는 림프구의 B세포에서 항체가 만들어질 때 특정항원에 의해 생성된 항체는 원칙적으로 다른 항원과 반응하지 않는다. 이러한 높은 특이성은 면역, 알레르기, 각종 병 및 감염의 종류·형의 결정 등의 검사에 사용된다.The term " antibody " is a protein produced by the stimulation of an antigen in the immune system, and is a protein that specifically binds to a specific antigen to migrate lymph and blood and cause an antigen-antibody reaction. The antigen-antibody reaction has a high specificity for each antigen. This is because antibodies produced by specific antigens do not react with other antigens in principle when antibodies are made in the lymphocyte B cells. Such high specificity is used in immunoassays, allergies, examination of various diseases, and determination of types and types of infections.
상기 용어 "항원"은 바이러스의 구성성분 중 면역기능을 일으킬 수 있는 성분으로서 바이러스가 발현하는 단백질이다. 본 발명의 일 실시예에서는, 일본 뇌염 바이러스 피막 단백질 돌출 도메인의 동형이량체 단백질이 개체에 투여되었을 때 면역 항체 형성을 유도하는 항원으로 작용하였다.The term " antigen " is a protein capable of inducing an immunological function among virus components and expressing the virus. In one embodiment of the present invention, when the homodimeric protein of the Japanese encephalitis virus coat protein protruding domain was administered to an individual, it acted as an antigen to induce immune antibody formation.
또한, 본 발명은 상기 동형이량체 단백질을 포함하는 일본 뇌염 바이러스 감염 진단용 키트를 제공하며, 상기 동형이량체 단백질에 특이적으로 결합하는 항체를 포함하는 일본 뇌염 바이러스 감염 진단용 키트를 제공한다.The present invention also provides a kit for the diagnosis of Japanese encephalitis virus infection comprising the homodimer protein, and a kit for diagnosing Japanese encephalitis virus infection comprising an antibody specifically binding to the homodimeric protein.
일 구체예로, 일본 뇌염 바이러스 피막 단백질 돌출 도메인의 동형이량체 단백질은 일본 뇌염 바이러스에 대한 항체와 특이적으로 결합할 수 있어, 일본 뇌염 바이러스 감염 진단 키트에 적용될 수 있다.In one embodiment, the homozygous protein of the Japanese encephalitis virus coat protein protruding domain can specifically bind to an antibody against Japanese encephalitis virus, and thus can be applied to a Japanese encephalitis virus infection diagnostic kit.
또한, 본 발명은 상기 일본 뇌염 바이러스 감염 진단용 키트에 진단 대상에서 분리된 시료를 첨가하는 단계를 포함하는 일본 뇌염 바이러스 감염 정보제공 방법을 제공한다.The present invention also provides a method for providing Japanese encephalitis virus infection information comprising the step of adding a sample isolated from a subject to a diagnostic kit for the Japanese encephalitis virus infection.
상기 용어 "일본 뇌염 바이러스 감염 진단용 키트"는 일본 뇌염 바이러스 감염 및 이에 의한 질병을 신속하게 진단할 수 있는 키트이다. 이러한 키트를 이용한 검사들은 10분 내지 15분 이내에 검사 결과를 알 수 있으며, 전문가 또는 진단을 위한 장비가 필요하지 않아 누구나 쉽게 일본 뇌염 바이러스 감염에 의한 질병을 진단할 수 있다는 장점이 있다.The term " kit for the diagnosis of Japanese encephalitis virus infection " is a kit capable of promptly diagnosing Japanese encephalitis virus infection and disease caused therefrom. Tests using these kits have an advantage of being able to diagnose diseases caused by Japanese encephalitis virus infection because experts can know the test results within 15 to 15 minutes and do not need equipment for experts or diagnosis.
본 발명은 또 다른 측면으로, 일본 뇌염 바이러스 피막 단백질 돌출 도메인 단량체 단백질을 코딩하는 뉴클레오티드를 포함하는 발현 벡터를 제공한다. 이때, 상기 벡터는 pAcGP67A-JEVE401일 수 있다.In another aspect, the present invention provides an expression vector comprising a nucleotide encoding a Japanese encephalitis virus coat protein protruding domain monomer protein. At this time, the vector may be pAcGP67A-JEVE401.
본 발명의 일 실시예에서는, 서열번호 3으로 표시되는 일본 뇌염 바이러스 피막 단백질의 둘출 도메인을 pAcGP67A 벡터의 BamHI과 ECoRI 부위에 서브클론하고 대장균 균주를 이용하여 과발현하였다. 이후, DNA midi-prep (Qiagen, 독일)을 이용하여 발현 벡터인 pAcGP67A-JEVE401를 정제하였다.In one embodiment of the present invention, the peritubular domain of the Japanese encephalitis virus coat protein represented by SEQ ID NO: 3 was subcloned into the BamHI and ECoRI sites of the pAcGP67A vector and overexpressed using E. coli strain. Then, the expression vector pAcGP67A-JEVE401 was purified using DNA midi-prep (Qiagen, Germany).
또한, 본 발명은 상기 발현 벡터로 형질감염된 숙주세포를 제공한다. 상기 숙주세포는 진핵세포일 수 있고, 상기 진핵세포는 곤충세포일 수 있다. 이때, 상기 숙주세포는 바큘로바이러스에 의해 추가로 형질감염될 수 있다.The present invention also provides a host cell transfected with said expression vector. The host cell may be a eukaryotic cell, and the eukaryotic cell may be an insect cell. At this time, the host cell may be further transfected with baculovirus.
본 발명의 일 실시예에서는, 형질감염 시약인 10% 프로펙틴(profectin)(AB vector 사)을 이용하여 상기 pAcGP67A-JEVE401 벡터와 바큘로 바이러스 DNA를 곤충세포인 sf9 세포에 형질감염시켰다.In one embodiment of the present invention, the pAcGP67A-JEVE401 vector and baculovirus DNA were transfected into insect cell sf9 cells using 10% profectin (AB vector) as a transfection reagent.
또한, 본 발명은 상기 형질감염된 숙주세포로부터 일본 뇌염 바이러스 피막 단백질 돌출 도메인 단량체 단백질을 수득하는 단계를 포함하는 일본 뇌염 바이러스 피막 단백질 돌출 도메인 단량체 단백질의 제조방법을 제공한다.The present invention also provides a method for producing a Japanese encephalitis virus coat protein protruding domain monomer protein, comprising the step of obtaining a Japanese encephalitis virus coat protein protruding domain monomer protein from the transfected host cell.
상기 수득된 일본 뇌염 바이러스 피막 단백질 돌출 도메인 단량체 단백질을 정제하는 단계를 포함할 수 있다. 이때, 상기 정제는 친화성 크로마토그래피 또는 젤 필트레이션 크로마토그래피 기법으로 수행될 수 있다.And purifying the obtained Japanese encephalitis virus coat protein protruding domain monomer protein. At this time, the purification can be performed by an affinity chromatography or gel filtration chromatography technique.
본 발명의 일 실시예에서는, 상기 pAcGP67A-JEVE401 벡터와 바큘로 바이러스 DNA로 형질감염된 곤충세포를 배양함으로써 일본 뇌염 바이러스 피막 단백질 돌출 도메인 단량체 단백질을 수득하였다.In one embodiment of the present invention, an insect cell transfected with the pAcGP67A-JEVE401 vector and baculovirus DNA was cultured to obtain a Japanese encephalitis virus coat protein protruding domain monomer protein.
본 발명은 또 다른 측면으로, (1) 상기 동형이량체 단백질을 항원으로서 실험동물에 주입하여 면역반응을 유도하는 단계; 및 (2) 상기 실험동물로부터 항체를 회수하는 단계를 포함하는 일본 뇌염 바이러스 항체 제조 방법을 제공한다.According to another aspect of the present invention, there is provided a method for producing an immunomodulatory composition comprising the steps of: (1) injecting an isoameric protein as an antigen into an experimental animal to induce an immune response; And (2) recovering the antibody from the laboratory animal.
이하, 본 발명을 하기 실시예에 의하여 더욱 상세하게 설명한다. 단, 하기 실시예는 본 발명을 예시하기 위한 것일 뿐, 본 발명의 범위가 이들만으로 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
실시예Example 1. One. 플라비Flavio 바이러스 및 일본 뇌염 바이러스의 피막 단백질 서열 비교분석 Comparative analysis of membrane protein sequences of virus and Japanese encephalitis virus
단백질 서열분석 비교 프로그램(Clustal W & ESPript 3.0)을 이용하여 플라비 바이러스 및 일본 뇌염 바이러스의 피막 단백질 아미노산 서열을 비교 분석하였다. 그 결과를 도 1 및 도 2에 나타내었다.Protein sequence analysis program (Clustal W & ESPript 3.0) was used to compare and analyze the amino acid sequences of Flavivirus and Japanese encephalitis virus. The results are shown in Fig. 1 and Fig.
도 1 및 도 2에 나타난 바와 같이, 플라비 바이러스 및 일본 뇌염 바이러스의 서열을 비교 분석하여 단백질 간의 보존된 서열을 확인하였고, 단백질의 구조를 기반으로 안정한 돌출도메인을 설계하였다.As shown in Fig. 1 and Fig. 2, the sequences of Flavivirus and Japanese encephalitis virus were compared and analyzed to confirm conserved sequences between proteins, and a stable protruding domain was designed based on the structure of proteins.
실시예Example 2. 2. 일본 뇌염 바이러스 피막 단백질 돌출도메인의 발현 벡터 제작 및 발현 확인Construction and expression confirmation of expression vector of Japanese encephalitis virus coat protein prominent domain
일본 뇌염 바이러스는 Nakayama strain(연세대학교)을 사용하였다. 상기 Nakayama strain의 피막 단백질은 바이러스에서 직접 유전자를 증폭하여 수득하였으며, 상기 피막 단백질의 염기 서열은 서열번호 1에 나타내었다.The Japanese encephalitis virus was the Nakayama strain (Yonsei University). The coat protein of the Nakayama strain was obtained by directly amplifying the gene in the virus, and the nucleotide sequence of the coat protein was shown in SEQ ID NO: 1.
일본 뇌염 바이러스 피막 단백질의 둘출 도메인을 pAcGP67A 벡터의 BamHI과 ECoRI 부위에 서브클로닝하고(subcloning)하고 대장균(Escherichia coli) DH10β 균주(strain)를 사용하여 벡터를 증폭하였다. 이후, DNA midi-prep (Qiagen, 독일)을 통하여 순수하게 발현 벡터(pAcGP67A-JEVE401)를 정제하였다. 형질감염 시약(10% profectin, AB vector 사)을 이용하여 정제한 pAcGP67A-JEVE401 벡터와 바큘로 바이러스 DNA를 곤충세포인 sf9 세포에 형질감염시켰다. 50시간 후, pAcGP67A-JEVE401을 포함한 바이러스 상층액을 수득하였다. 이 상층액을 1:20의 비율로 다시 감염시키는 방식으로 5회 계대 배양한 후, 배양 상층액으로 분비된 피막 단백질의 돌출 도메인 발현을 웨스턴 블롯을 이용하여 확인하였다. 그 결과를 도 3에 나타내었다.The pertussis domain of the Japanese encephalitis virus coat protein was subcloned into the BamHI and ECoRI sites of the pAcGP67A vector and Escherichia coli The vector was amplified using E. coli DH10 [beta] strain. Subsequently, a pure expression vector (pAcGP67A-JEVE401) was purified through DNA midi-prep (Qiagen, Germany). PAcGP67A-JEVE401 vector and baculovirus DNA purified using a transfection reagent (10% profectin, AB vector) were transfected into insect cell sf9 cells. After 50 hours, a virus supernatant containing pAcGP67A-JEVE401 was obtained. The supernatant was re-infected at a ratio of 1: 20, followed by subculture for 5 times, and then the expression of the protruding domain of the membrane protein secreted into the culture supernatant was confirmed by Western blotting. The results are shown in Fig.
실시예Example 3. 일본 뇌염 바이러스 피막 단백질 돌출도메인의 대량 발현 3. Mass expression of Japanese encephalitis virus coat protein prominent domains
상기 실시예 2에서 확인한 일본 뇌염 바이러스 피막 단백질 돌출 도메인의 대량 발현을 위하여 2.5% FBS와 함께 배양한 곤충세포 Hi5 세포의 배양액을 1.5 X 106 cell/ml 내지 2.0 X 106 cell/ml까지 배양하였다. 이후, 상기 배양액을 1:20의 비율로 바이러스 감염시키고 27℃에서 4일 동안 배양하였다. 배양 후, 상기 배양액의 상층액을 원심분리하여 일본 뇌염 바이러스 피막 단백질의 돌출 도메인을 수득하였다.For the mass expression of the Japanese encephalitis virus coat protein protruding domain identified in Example 2, a culture medium of insect cell Hi5 cells cultured with 2.5% FBS was cultured to 1.5 x 10 6 cells / ml to 2.0 x 10 6 cells / ml . Then, the culture solution was viral infected at a ratio of 1:20 and cultured at 27 DEG C for 4 days. After the culture, the supernatant of the culture was centrifuged to obtain a protruding domain of the Japanese encephalitis virus coat protein.
실시예Example 4. 일본 뇌염 바이러스 피막 단백질 돌출 도메인의 정제 4. Purification of Japanese encephalitis virus coat protein extrusion domain
6개의 히스티딘이 표지된 일본 뇌염 바이러스 피막 단백질 돌출 도메인의 Ni-NTA를 이용한 친화 크로마토그래피법 정제를 위하여, 컬럼 평형화 완충 용액인 pH 7.5의 20 mM Tris-HCl 용액과 0.2 M NaCl 용액으로 3일 동안 투석(dialysis) 과정을 수행하였다. 투석 후, 상기 피막 단백질 돌출 도메인이 포함된 상층액을 Ni-NTA(nickel nitrilotriacetic acid) 아가로오스(agarose) 컬럼에 천천히 결합시켰다. 이후, 상기와 동일한 완충 용액으로 컬럼을 세척하고, 0.5 M 이미다졸(imidazole)이 포함된 완충 용액을 이용하여 선형농도구배법으로 단백질을 용출하면서 분획을 모았다. 이후, SDS-PAGE를 통하여 용출된 단백질을 확인하였다. 그 결과를 도 4a 및 도 4b에 나타내었다.For purification of affinity chromatography using 6-histidine-labeled Japanese encephalitis virus coat protein proliferation domain using Ni-NTA, a 20 mM Tris-HCl solution of pH 7.5, a column equilibration buffer solution, and a 0.2 M NaCl solution were added for 3 days A dialysis process was performed. After dialysis, the supernatant containing the above-mentioned membrane protein protruding domains was slowly bound to an Ni-NTA (nickel nitrilotriacetic acid) agarose column. Thereafter, the column was washed with the same buffer solution as above, and the fractions were collected by eluting proteins using a linear concentration gradient method using a buffer solution containing 0.5 M imidazole. Thereafter, the eluted protein was confirmed by SDS-PAGE. The results are shown in Figs. 4A and 4B.
상기 확인된 단백질은 농축기(Vivaspin, MWCO 30KDa)를 통하여 5 ml까지 농축한 후, 2배 농도의 PBS (Phosphate buffered saline) 완충 용액으로 평형화시킨 Superdex 200 (GE Healthcare, 16X600) 젤 필트레이션(Gel Filtration) 크로마토그래피법을 이용하여 최종 정제하였다. 상기 정제된 단백질은 Bradford assay (Bio-rad사)법으로 농도를 정량한 후 1 mg/ml로 농축하여 표준 단백질인 1 mg/ml의 소혈청 알부민과 SDS-PAGE 상에서 비교하였다. 그 결과를 도 4c 내지 도 4d에 나타내었다. 또한, 상기 정제된 일본 뇌염 바이러스 피막 단백질 돌출도메인의 발현을 SDS 상에서 확인한 것을 도 4e에 나타내었다. 이후, 상기 정제된 단백질을 분주하여 -80℃에서 실험 전까지 보관하였다.The identified proteins were concentrated to 5 ml using Vivaspin (MWCO 30KDa), and then purified using Superdex 200 (GE Healthcare, 16X600) gel filtration (PBS), which was equilibrated with 2x concentration of phosphate buffered saline buffer ) Chromatography. The purified protein was quantitated by Bradford assay (Bio-rad), concentrated to 1 mg / ml and compared with SDS-PAGE with 1 mg / ml bovine serum albumin. The results are shown in Figs. 4C to 4D. In addition, the expression of the purified Japanese encephalitis virus coat protein overexpression domain on SDS was shown in Fig. 4E. Thereafter, the purified protein was dispensed and stored at -80 캜 until the experiment.
도 4a 내지 도 4e에 나타난 바와 같이, 상기 실시예 1에서 제작한 일본 뇌염 바이러스 피막 단백질 돌출 도메인은 Ni-NTA 친화성 크로마토그래피 및 젤 필트레이션 크로마토그래피 기법에 의한 정제 과정을 통해 순도 98% 이상 정제되었다.As shown in Figs. 4A to 4E, the Japanese encephalitis virus coat protein protruding domain prepared in Example 1 was purified through Ni-NTA affinity chromatography and gel filtration chromatography to a purity of 98% or more .
실시예Example 5. 항체 생성 실험 5. Antibody production experiment
상기 실시예 4의 정제 과정을 통해 수득된 일본 뇌염 바이러스 피막 단백질의 돌출 도메인의 동형이량체 단백질 항원에 대한 항체 생성 실험을 진행하였다. 먼저, 상기 동형이량체 단백질 항원을 마우스에 주사하기 전, pre-immune serum을 채취하여 음성 대조군으로 사용하였다. 1차 면역(primary immunization)은 항원 200 ㎍과 혼합하여 복강(intraperitoneal, IP) 주사하였고, 2주 후 불완전한 프로이드 어주번트(incomplete freund's adjuvant, IFA)(sigma)와 혼합하여 1차 부스팅(boosting)을 진행하였다. 1주 후, 1차 혈액을 채취하여 ELISA 테스트를 진행하였다. 1주 간격으로 부스팅과 블리딩을 진행하였다. 3차 부스팅을 진행하고 1주 후, 마우스의 심장으로부터 혈액을 채취하고 최종 혈청을 분리하여 최종 ELISA 테스트를 진행하였다. ELISA 테스트 시 항원은 100 ng/well로 코팅하여 사용하였다. 또한, 각 부스팅 시기별로 채혈하여 보관한 혈청은 0.2% skim milk를 포함한 0.05% tween PBS용액(이하 0.2% skim milk)을 이용하여 일정 비율 희석하여 사용하였으며, 1:10,000으로 희석한 Anti-mouse IgG-HRP(ABC5001)를 이용하여 항원특이적 항체를 검출하였다.The antibody production test for the homologous protein antigen of the protruding domain of the Japanese encephalitis virus coat protein obtained through the purification process of Example 4 was carried out. Prior to injecting the homodimeric protein antigen into mice, pre-immune serum was collected and used as a negative control. Primary immunization was performed by intraperitoneal (IP) injection with 200 μg of antigen. After two weeks, primary incomplete freund's adjuvant (IFA) (Sigma) was mixed with primary boosting . One week later, primary blood was collected and subjected to ELISA test. Boosting and bleeding were performed at intervals of one week. One week after the third booster, blood was collected from the mouse heart and the final serum was separated to conduct the final ELISA test. In the ELISA test, the antigen was coated at 100 ng / well. In addition, serum collected and stored at each boosting period was diluted with 0.05% tween PBS solution containing 0.2% skim milk (hereinafter referred to as 0.2% skim milk) at a ratio of 1: 10,000 and diluted with anti-mouse IgG -HRP (ABC5001) was used to detect the antigen-specific antibody.
실험예Experimental Example 1. 일본 뇌염 바이러스 피막 단백질의 돌출 도메인의 1. Japanese encephalitis viral coat protein prominent domain 동형이량체Homodimer 단백질 항원의 면역 항체 형성 실험 Immuno-antibody formation experiment of protein antigen
1.1. 항체 형성 실험1.1. Antibody formation experiment
일본 뇌염 바이러스 피막 단백질의 돌출 도메인의 동형이량체 단백질 항원의 면역 항체 형성을 확인하기 위하여, 상기 실시예 4에서 정제된 일본 뇌염 바이러스 피막 단백질의 돌출 도메인의 동형이량체 단백질 항원(JEP401)을 4마리의 마우스에 면역하였다. 4주 후, 각 마우스로부터 최종 혈청을 분리하여 ELISA 테스트를 진행하였다. ELISA 테스트 시, 상기 항원은 웰 당 100 ng을 사용하였으며, 상기 분리한 면역 혈청은 1X PBS로 1:100 내지 1:100,000 비율로 희석하였다. 또한, 2차 확인을 위하여 항-마우스 IgG-HRP (ABC5001)을 사용하였으며, 405 nm의 흡광도(Optical Density, OD)에서 발색을 검출하여 비교하였다. 이를 표 1 및 도 5에 나타내었다.To confirm immune antibody formation of the homozygous protein antigen of the protruding domain of the Japanese encephalitis virus coat protein, 4 mice (JEP401) of the protruding domain of the Japanese encephalitis virus coat protein purified in Example 4 above Lt; / RTI > mice. After 4 weeks, the final serum was separated from each mouse and subjected to an ELISA test. In the ELISA test, the antigen was used at 100 ng per well, and the separated immunized serum was diluted with 1X PBS at a ratio of 1: 100 to 1: 100,000. In addition, anti-mouse IgG-HRP (ABC5001) was used for the secondary identification, and color development was detected at 405 nm optical density (OD). This is shown in Table 1 and FIG.
Test serum
Test serum
표 1 및 도 5에 나타난 바와 같이, 일본 뇌염 바이러스 피막 단백질의 돌출 도메인의 동형이량체 단백질 항원을 주입한 마우스에서 항체 농도가 증가하였다. 이를 통해, 일본 뇌염 바이러스 피막 단백질의 돌출 도메인의 동형이량체 단백질 항원을 개체에 주입할 경우, 이에 대한 면역 항체가 잘 생성되어 백신으로써 효능이 있음을 알 수 있었다.As shown in Table 1 and Fig. 5, the antibody concentration increased in mice injected with homozygous protein antigen in the protruding domain of the Japanese encephalitis virus coat protein. Thus, when the homozygous protein antigen of the protruding domain of the Japanese encephalitis virus coat protein is injected into the individual, the immune antibody is well formed and the vaccine is effective.
1.2. 일본 뇌염 바이러스 피막 단백질의 돌출 도메인의 1.2. Of the protruding domain of the Japanese encephalitis virus coat protein 동형이량체Homodimer 단백질 항원의 면역 횟수에 따른 항체 형성 차이 확인 Identification of Antibody Formation by Protein Antigen Counts
일본 뇌염 바이러스 피막 단백질의 돌출 도메인의 동형이량체 단백질 항원의 면역 횟수에 따른 항체 형성 차이를 확인하기 위하여, 상기 실시예 4에서 정제된 일본 뇌염 바이러스 피막 단백질의 돌출 도메인의 동형이량체 단백질 항원을 2마리의 마우스에게 각각 2번 및 3번 면역하였다. 이후, ELISA 기법을 이용하여 상기 각 마우스의 혈청에 존재하는 일본 뇌염 바이러스의 항체를 측정하였다. 그 결과를 도 6에 나타내었다.In order to confirm the difference in antibody formation between the protruding domain of the Japanese encephalitis virus coat protein and the immunogenicity of the homodimeric protein antigen, the homozygous protein antigen of the protruding domain of the Japanese encephalitis virus coat protein purified in Example 4 was labeled with 2 The mice were immunized twice and three times, respectively. Then, the antibody of Japanese encephalitis virus present in the serum of each mouse was measured using an ELISA technique. The results are shown in Fig.
도 6에 나타난 바와 같이, 상기 항원을 3번 면역한 마우스(JEVx3)의 혈액 속 항체의 농도는 상기 항원을 2번 면역한 마우스(JEVx2)에 비해 높게 나타났다. 이는 상기 돌출 도메인 항원의 투여 횟수가 증가할수록, 이의 항체 형성이 증가함을 의미한다. As shown in FIG. 6, the concentration of the antibody in the blood of the mouse (JEVx3) immunized with the antigen three times higher than that of the mouse (JEVx2) immunized with the antigen twice. This means that as the number of administration of the protruding domain antigens is increased, its antibody formation is increased.
1.3. 항체 형성 클론 세포 확인1.3. Identifying antibody-forming clonal cells
일본 뇌염 바이러스 피막 단백질의 돌출 도메인의 동형이량체 단백질 항원의 항체를 형성하는 클론 세포를 확인하기 위해, 상기 실험예 1.1의 방법에 따라 일본 뇌염 바이러스 피막 단백질의 돌출 도메인의 동형이량체 단백질 항원의 단클론 항체를 제작하여 ELISA 테스트 및 아이소타이핑 스크리닝(isotyping screening)을 진행하였다. 이를 표 2 및 표 3에 나타내었다.In order to identify clone cells that form an antibody of the homozygous protein antigen of the protruding domain of the Japanese encephalitis virus coat protein, the monoclonal antibody of the homologous protein antigen of the protruding domain of the Japanese encephalitis virus coat protein according to the method of Experimental Example 1.1 Antibodies were prepared and subjected to ELISA testing and isotyping screening. These are shown in Tables 2 and 3.
실험예Experimental Example 2. 곤충세포 유래 일본 뇌염 바이러스 피막 단백질의 돌출 도메인의 동형이량체 단백질 항원의 특이도 확인 2. Identification of homozygous protein antigen of the protruding domain of Japanese encephalitis virus coat protein derived from insect cells
본 발명에 따른 곤충세포 유래 일본 뇌염 바이러스 피막 단백질의 돌출 도메인의 동형이량체 단백질 항원과 대장균 발현 시스템 유래 항원의 특이도를 비교하기 위하여, 고농도의 혈청(1:25)과 항원(200, 400, 800 ng/well)을 사용하여 ELISA 테스트를 실시하였다. 그 결과를 도 7에 나타내었다.In order to compare the specificity of the homologous protein antigen and the antigens derived from the Escherichia coli expression system in the protruding domain of the insect cell-derived Japanese encephalitis virus coat protein according to the present invention, high serum (1:25) and antigen (200, 400, 800 ng / well) was used for the ELISA test. The results are shown in Fig.
도 7에 나타난 바와 같이, 대장균 발현 시스템 유래 항원(LysRS-JEV-E)에서는 항원-항체 반응이 거의 검출되지 않았다. 반면, 곤충세포 유래 일본 뇌염 바이러스 피막 단백질의 돌출 도메인의 동형이량체 단백질 항원(JEV-E)에서는 항원-항체 반응이 높게 나타났다. 이는, 본 발명의 곤충세포 유래 항원이 대장균 시스템 유래 항원보다 민감도 및 특이도가 월등히 우수함을 의미한다.As shown in Fig. 7, almost no antigen-antibody reaction was detected in the E. coli expression system-derived antigen (LysRS-JEV-E). On the other hand, the homozygous protein antigen (JEV-E) of the protruding domain of the insect cell-derived Japanese encephalitis virus coat protein showed a high antigen-antibody reaction. This means that the insect cell-derived antigen of the present invention is far superior in sensitivity and specificity than the E. coli system-derived antigen.
실험예Experimental Example 3: 일본 뇌염 바이러스 피막 단백질의 돌출 도메인의 3: Prominent domain of Japanese encephalitis virus coat protein 동형이량체Homodimer 백신 효능 평가 Evaluation of vaccine efficacy
본 발명에 따른 동형이량체 단백질이 실제 혈청 내 항체가 측정에 적용될 수 있는지 여부를 확인하기 위해, 일본 뇌염 양성 마우스 혈청을 제작하여 확보하였다. ELISA 기법에 대한 정제 단백의 효용성 및 실험기법의 민감도 및 정확도를 비교하기 위하여, 지금까지 일본 뇌염 면역원성 평가의 최적 표준으로 이용되어온 PRNT(plaque reduction neutralization test) 기법의 SOP (Shirley H.L et al., Journal of Virological Methods, 2001)를 구축하였다. 마우스 유래 일본 뇌염 양성 항혈청을 이용하여, 새로이 개발된 ELISA 기법과 PRNT 검증법과의 비교분석을 수행하였다. 그 결과를 표 4, 도 8a, 및 도 8b에 나타내었다.In order to confirm whether or not the homodimeric protein of the present invention can be applied to the actual serum antibody, Japanese encephalitis-positive mouse serum was prepared and secured. In order to compare the sensitivity and accuracy of the purified protein to the ELISA technique and the accuracy of the experimental technique, the SOP of the PRNT (plaque reduction neutralization test) method ( Shirley HL et al. Journal of Virological Methods, 2001 ). We performed comparative analysis with the newly developed ELISA technique and PRNT assays using mouse derived Japanese encephalitis positive antiserum. The results are shown in Tables 4, 8A and 8B.
SA14-14-2
SA 14 -14-2
MVA(Vjh6)
MVA (Vjh6)
이때, 표 4는 항원과 면역증강제를 설하투여와 근육주사방법으로 투여하여 얻은 혈청을 이용하여 TCID50값과 항체의 중화능력을 titer로 나타난 값이다.Table 4 shows the titer of the TCID 50 value and neutralizing ability of the antibody using sera obtained by administration of the antigen and the immunostimulant by sublingual administration and intramuscular injection.
도 8a는 상기 실험에서 얻은 혈청을 이용하여 ELISA를 통해 각 샘플의 항JEV혈청의 양을 측정한 값으로서, 혈청을 1:100에서 1:10,000까지 순차적으로 희석하여 혈청속에 있는 항체의 정도를 측정한 것이다.FIG. 8A is a graph showing the amount of anti-JEV serum in each sample measured by ELISA using the serum obtained in the above experiment. Serum was sequentially diluted from 1: 100 to 1: 10,000 to measure the level of the antibody in the serum It is.
도 8b는 도 8a와 같은 혈청 샘플을 이용하여 그 속에 있는 JEV항체의 농도를 마우스 표준 IgG를 이용하여 정량화 한 값이다. 두 가지 면역 증강제 SA-14-2와 MAV를 비교하였을 때 MAV가 더 좋은 효과를 보이는 것은 알 수 있었고 설하(sublingual, s.l.) 백신보다 근육(intramuscular, i.m.)으로 백신을 투여한 경우가 더 효과가 좋으나 MAV의 경우 설하백신도 충분히 좋은 백신의 효과를 나타내도록 면역증강제로서의 역할을 한다는 것을 정량적으로 알 수 있었다. 이 실험은 혈액 속의 JEV항체가를 측정할 수 있을 뿐만 아니라 백신개발이나 치료제 개발과정에서도 진단기법으로 쉽게 사용할 수 있을 것으로 보인다. FIG. 8B is a value obtained by quantifying the concentration of the JEV antibody in the serum sample using the mouse standard IgG using the serum sample as shown in FIG. 8A. Compared with two MAVs, MAV showed better efficacy, and vaccination with intramuscular (im) rather than sublingual (sl) vaccine was more effective But it was quantitatively demonstrated that MAV vaccine also plays a role as an immunity enhancer to show a sufficiently good vaccine effect. In addition to being able to measure JEV antibody titers in the blood, this experiment can be easily used as a diagnostic tool in the development of vaccines or in the development of therapeutic agents.
표 4, 도 8a, 및 도 8b에 나타난 바와 같이, JEV 백신 후보물질을 투여한 마우스의 혈청샘플에서 유의미한 값의 anti-JEV IgG가 측정되었고, 이 값은 TCID50 및 PRNT 분석을 통해 도출된 neutralizing antibody 측정값과 같은 양상을 나타내었다. 이는 새로이 확립된 ELISA 기반 항체가 측정 기법이 일본 뇌염 표준 항체가 측정법으로 사용 가능한 수준의 신뢰도를 가짐을 의미한다. 특히, 본 실험에서 수행한 ELISA 기법은 기존의 PRNT 기법에 비해 실험과정이 단순하고 살아있는 바이러스를 사용하지 않아 안전할 뿐 아니라 특별한 실험시설을 갖춘 곳에서 진행하지 않아도 일본뇌염 바이러스의 항체의 정량적 값을 도출할 수 있어, 연구 및 임상에서 활용성이 매우 높을 것으로 기대된다.Table 4, Figure 8a, and as shown in Figure 8b, were the anti-JEV IgG of significant value measured in serum samples of the mice treated with JEV vaccine candidates, the value neutralizing derived from the TCID 50 and PRNT analysis antibody. This means that the newly established ELISA-based antibody measurement technique has a level of confidence that the Japanese encephalitis standard antibody can be used as a measurement method . In particular, the ELISA method performed in this experiment is safe because it is simple and does not use living viruses as compared with the conventional PRNT method. Moreover, it does not require the use of a special laboratory facility and the quantitative value of the antibody of Japanese encephalitis virus And it is expected to be very useful in research and clinical applications.
<110> Korea Research Institute of Bioscience and Biotechnology Industry-Academic Cooperation Foundation, Yonsei University <120> PROTEIN COMPRISING ECTODOMAIN OF JAPANESE ENCEPHALITIS VIRUS ENVELOPE PROTEINS, HOMODIMER THEREOF, AND USE THEREOF <130> FPD/201708-0036 <160> 11 <170> KoPatentIn 3.0 <210> 1 <211> 1500 <212> DNA <213> Artificial Sequence <220> <223> Nucleic acid sequence for Japanese encephalitis virus envelop protein (strain: Nakayama) <400> 1 ttcaactgtc tgggaatggg caatcgtgac ttcatagaag gagccagtgg agccacttgg 60 gtggacttgg tgctagaagg agacagctgc ttgacaatta tggcaaacga caaaccaaca 120 ttggacgtcc gcatgatcaa catcgaagct gtccaacttg ctgaggtcag aagttactgc 180 tatcatgctt cagtcactga catttcgacg gtggctcggt gccccacgac tggagaagct 240 cacaacaaga agcgagctga tagtagctat gtgtgcaaac aaggcttcac tgatcgtggg 300 tggggcaacg gatgtggact tttcgggaag ggaagcattg acacatgtgc aaaattctcc 360 tgcaccagta aggcgattgg gagaacaatc cagccagaaa acatcaaata cgaagttggc 420 atttttgtgc atggaaccac cacttcggaa aaccatggga attattcagc gcaagttggg 480 gcgtcccagg cggcaaagtt tacagtaaca cccaatgctc cttcgataac cctcaaactt 540 ggtgactacg gagaagtcac actggactgt gagccaagga gtggactaaa cactgaagcg 600 ttttacgtca tgaccgtggg gtcaaagtca tttttggtcc acagggaatg gtttcatgat 660 ctcgctctcc cttggacgcc cccttcgagc acagcgtgga gaaacagaga actcctcatg 720 gaatttgaag aggcgcacgc cacaaaacag tccgttgttg ctcttgggtc acaggaagga 780 ggcctccatc aggcgttggc aggagccatc gtggtggagt actcaaactc agtgaagtta 840 acatcaggcc acctaaaatg caggctgaga atggacaaac tggctctgaa aggcacaacc 900 tatggcatgt gcacagaaaa attctcgttc gcgaaaaatc cggcggacac tggtcacgga 960 acagttgtca ttgaactttc ctactctggg agtgatggcc cttgcaaaat tccgattgtc 1020 tccgttgcga gcctcaatga catgaccccc gtcgggcggc tggtgacagt gaaccccttc 1080 gtcgcgactt ccagcgccaa ctcaaaggtg ctagtcgaga tggaaccccc cttcggagac 1140 tcctacatcg tagttggaag gggagacaag cagattaacc accattggca caaggctgga 1200 agcacgctgg gcaaagcctt ttcaacgact ttgaagggag ctcaaagact ggtagcgttg 1260 ggcgacacag cctgggactt tggctctatt ggaggggttt tcaactccat agggaaagcc 1320 gttcaccaag tgtttggtgg tgccttcaga acactcttcg ggggaatgtc ttggatcaca 1380 caagggctaa tgggggccct actactctgg atgggcgtca acgcacgaga ccgatcaatt 1440 gctttggcct tcttagccac aggaggtgtg ctcgtgttct tagcgaccaa tgtgcatgct 1500 1500 <210> 2 <211> 1203 <212> DNA <213> Artificial Sequence <220> <223> Nucleic acid sequence for ectodomain of Japanese encephalitis virus envelop protein (strain: Nakayama) <400> 2 ttcaactgtc tgggaatggg caatcgtgac ttcatagaag gagccagtgg agccacttgg 60 gtggacttgg tgctagaagg agacagctgc ttgacaatta tggcaaacga caaaccaaca 120 ttggacgtcc gcatgatcaa catcgaagct gtccaacttg ctgaggtcag aagttactgc 180 tatcatgctt cagtcactga catttcgacg gtggctcggt gccccacgac tggagaagct 240 cacaacaaga agcgagctga tagtagctat gtgtgcaaac aaggcttcac tgatcgtggg 300 tggggcaacg gatgtggact tttcgggaag ggaagcattg acacatgtgc aaaattctcc 360 tgcaccagta aggcgattgg gagaacaatc cagccagaaa acatcaaata cgaagttggc 420 atttttgtgc atggaaccac cacttcggaa aaccatggga attattcagc gcaagttggg 480 gcgtcccagg cggcaaagtt tacagtaaca cccaatgctc cttcgataac cctcaaactt 540 ggtgactacg gagaagtcac actggactgt gagccaagga gtggactaaa cactgaagcg 600 ttttacgtca tgaccgtggg gtcaaagtca tttttggtcc acagggaatg gtttcatgat 660 ctcgctctcc cttggacgcc cccttcgagc acagcgtgga gaaacagaga actcctcatg 720 gaatttgaag aggcgcacgc cacaaaacag tccgttgttg ctcttgggtc acaggaagga 780 ggcctccatc aggcgttggc aggagccatc gtggtggagt actcaaactc agtgaagtta 840 acatcaggcc acctaaaatg caggctgaga atggacaaac tggctctgaa aggcacaacc 900 tatggcatgt gcacagaaaa attctcgttc gcgaaaaatc cggcggacac tggtcacgga 960 acagttgtca ttgaactttc ctactctggg agtgatggcc cttgcaaaat tccgattgtc 1020 tccgttgcga gcctcaatga catgaccccc gtcgggcggc tggtgacagt gaaccccttc 1080 gtcgcgactt ccagcgccaa ctcaaaggtg ctagtcgaga tggaaccccc cttcggagac 1140 tcctacatcg tagttggaag gggagacaag cagattaacc accattggca caaggctgga 1200 agc 1203 <210> 3 <211> 411 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence for ectodomain of Japanese encephalitis virus envelop protein (strain: Nakayama) <400> 3 Gly Ser Phe Asn Cys Leu Gly Met Gly Asn Arg Asp Phe Ile Glu Gly 1 5 10 15 Ala Ser Gly Ala Thr Trp Val Asp Leu Val Leu Glu Gly Asp Ser Cys 20 25 30 Leu Thr Ile Met Ala Asn Asp Lys Pro Thr Leu Asp Val Arg Met Ile 35 40 45 Asn Ile Glu Ala Val Gln Leu Ala Glu Val Arg Ser Tyr Cys Tyr His 50 55 60 Ala Ser Val Thr Asp Ile Ser Thr Val Ala Arg Cys Pro Thr Thr Gly 65 70 75 80 Glu Ala His Asn Lys Lys Arg Ala Asp Ser Ser Tyr Val Cys Lys Gln 85 90 95 Gly Phe Thr Asp Arg Gly Trp Gly Asn Gly Cys Gly Leu Phe Gly Lys 100 105 110 Gly Ser Ile Asp Thr Cys Ala Lys Phe Ser Cys Thr Ser Lys Ala Ile 115 120 125 Gly Arg Thr Ile Gln Pro Glu Asn Ile Lys Tyr Glu Val Gly Ile Phe 130 135 140 Val His Gly Thr Thr Thr Ser Glu Asn His Gly Asn Tyr Ser Ala Gln 145 150 155 160 Val Gly Ala Ser Gln Ala Ala Lys Phe Thr Val Thr Pro Asn Ala Pro 165 170 175 Ser Ile Thr Leu Lys Leu Gly Asp Tyr Gly Glu Val Thr Leu Asp Cys 180 185 190 Glu Pro Arg Ser Gly Leu Asn Thr Glu Ala Phe Tyr Val Met Thr Val 195 200 205 Gly Ser Lys Ser Phe Leu Val His Arg Glu Trp Phe His Asp Leu Ala 210 215 220 Leu Pro Trp Thr Pro Pro Ser Ser Thr Ala Trp Arg Asn Arg Glu Leu 225 230 235 240 Leu Met Glu Phe Glu Glu Ala His Ala Thr Lys Gln Ser Val Val Ala 245 250 255 Leu Gly Ser Gln Glu Gly Gly Leu His Gln Ala Leu Ala Gly Ala Ile 260 265 270 Val Val Glu Tyr Ser Asn Ser Val Lys Leu Thr Ser Gly His Leu Lys 275 280 285 Cys Arg Leu Arg Met Asp Lys Leu Ala Leu Lys Gly Thr Thr Tyr Gly 290 295 300 Met Cys Thr Glu Lys Phe Ser Phe Ala Lys Asn Pro Ala Asp Thr Gly 305 310 315 320 His Gly Thr Val Val Ile Glu Leu Ser Tyr Ser Gly Ser Asp Gly Pro 325 330 335 Cys Lys Ile Pro Ile Val Ser Val Ala Ser Leu Asn Asp Met Thr Pro 340 345 350 Val Gly Arg Leu Val Thr Val Asn Pro Phe Val Ala Thr Ser Ser Ala 355 360 365 Asn Ser Lys Val Leu Val Glu Met Glu Pro Pro Phe Gly Asp Ser Tyr 370 375 380 Ile Val Val Gly Arg Gly Asp Lys Gln Ile Asn His His Trp His Lys 385 390 395 400 Ala Gly Ser Leu Glu His His His His His His 405 410 <210> 4 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Binding site on ectodomain of Japanese encephalitis virus envelop protein <400> 4 Phe Asn Cys Leu Gly Met Gly Asn Arg Asp 1 5 10 <210> 5 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Binding site on ectodomain of Japanese encephalitis virus envelop protein <400> 5 Val Leu Glu Gly Asp Ser Cys Leu Thr 1 5 <210> 6 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Binding site on ectodomain of Japanese encephalitis virus envelop protein <400> 6 His Ala Ser Val Thr Asp Ile Ser 1 5 <210> 7 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Binding site on ectodomain of Japanese encephalitis virus envelop protein <400> 7 Gly Trp Gly Asn Gly Cys Gly Leu 1 5 <210> 8 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Binding site on ectodomain of Japanese encephalitis virus envelop protein <400> 8 Thr Thr Thr Ser Glu Asn His Gly Asn 1 5 <210> 9 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Binding site on ectodomain of Japanese encephalitis virus envelop protein <400> 9 Ala His Ala Thr Lys Gln Ser Val Val Ala 1 5 10 <210> 10 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Binding site on ectodomain of Japanese encephalitis virus envelop protein <400> 10 Gln Glu Gly Gly Leu His Gln Ala Leu Ala Gly Ala Ile Val Val Glu 1 5 10 15 Tyr Ser <210> 11 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Binding site on ectodomain of Japanese encephalitis virus envelop <400> 11 Asp Thr Gly His Gly Thr Val 1 5 <110> Korea Research Institute of Bioscience and Biotechnology Industry-Academic Cooperation Foundation, Yonsei University <120> PROTEIN COMPRISING ECTODOMAIN OF JAPANESE ENCEPHALITIS VIRUS ENVELOPE PROTEINS, HOMODIMER THEREOF, AND USE THEREOF <130> FPD / 201708-0036 <160> 11 <170> KoPatentin 3.0 <210> 1 <211> 1500 <212> DNA <213> Artificial Sequence <220> <223> Nucleic acid sequence for Japanese encephalitis virus envelope protein (strain: Nakayama) <400> 1 ttcaactgtc tgggaatggg caatcgtgac ttcatagaag gagccagtgg agccacttgg 60 gtggacttgg tgctagaagg agacagctgc ttgacaatta tggcaaacga caaaccaaca 120 ttggacgtcc gcatgatcaa catcgaagct gtccaacttg ctgaggtcag aagttactgc 180 tatcatgctt cagtcactga catttcgacg gtggctcggt gccccacgac tggagaagct 240 cacaacaaga agcgagctga tagtagctat gtgtgcaaac aaggcttcac tgatcgtggg 300 tggggcaacg gatgtggact tttcgggaag ggaagcattg acacatgtgc aaaattctcc 360 tgcaccagta aggcgattgg gagaacaatc cagccagaaa acatcaaata cgaagttggc 420 atttttgtgc atggaaccac cacttcggaa aaccatggga attattcagc gcaagttggg 480 gcgtcccagg cggcaaagtt tacagtaaca cccaatgctc cttcgataac cctcaaactt 540 ggtgactacg gagaagtcac actggactgt gagccaagga gtggactaaa cactgaagcg 600 ttttacgtca tgaccgtggg gtcaaagtca tttttggtcc acagggaatg gtttcatgat 660 ctcgctctcc cttggacgcc cccttcgagc acagcgtgga gaaacagaga actcctcatg 720 gaatttgaag aggcgcacgc cacaaaacag tccgttgttg ctcttgggtc acaggaagga 780 ggcctccatc aggcgttggc aggagccatc gtggtggagt actcaaactc agtgaagtta 840 acatcaggcc acctaaaatg caggctgaga atggacaaac tggctctgaa aggcacaacc 900 tatggcatgt gcacagaaaa attctcgttc gcgaaaaatc cggcggacac tggtcacgga 960 acagttgtca ttgaactttc ctactctggg agtgatggcc cttgcaaaat tccgattgtc 1020 tccgttgcga gcctcaatga catgaccccc gtcgggcggc tggtgacagt gaaccccttc 1080 gtcgcgactt ccagcgccaa ctcaaaggtg ctagtcgaga tggaaccccc cttcggagac 1140 tcctacatcg tagttggaag gggagacaag cagattaacc accattggca caaggctgga 1200 agcacgctgg gcaaagcctt ttcaacgact ttgaagggag ctcaaagact ggtagcgttg 1260 ggcgacacag cctgggactt tggctctatt ggaggggttt tcaactccat agggaaagcc 1320 gttcaccaag tgtttggtgg tgccttcaga acactcttcg ggggaatgtc ttggatcaca 1380 caagggctaa tgggggccct actactctgg atgggcgtca acgcacgaga ccgatcaatt 1440 gctttggcct tcttagccac aggaggtgtg ctcgtgttct tagcgaccaa tgtgcatgct 1500 1500 <210> 2 <211> 1203 <212> DNA <213> Artificial Sequence <220> <223> Nucleic acid sequence for ectodomain of Japanese encephalitis virus envelop protein (strain: Nakayama) <400> 2 ttcaactgtc tgggaatggg caatcgtgac ttcatagaag gagccagtgg agccacttgg 60 gtggacttgg tgctagaagg agacagctgc ttgacaatta tggcaaacga caaaccaaca 120 ttggacgtcc gcatgatcaa catcgaagct gtccaacttg ctgaggtcag aagttactgc 180 tatcatgctt cagtcactga catttcgacg gtggctcggt gccccacgac tggagaagct 240 cacaacaaga agcgagctga tagtagctat gtgtgcaaac aaggcttcac tgatcgtggg 300 tggggcaacg gatgtggact tttcgggaag ggaagcattg acacatgtgc aaaattctcc 360 tgcaccagta aggcgattgg gagaacaatc cagccagaaa acatcaaata cgaagttggc 420 atttttgtgc atggaaccac cacttcggaa aaccatggga attattcagc gcaagttggg 480 gcgtcccagg cggcaaagtt tacagtaaca cccaatgctc cttcgataac cctcaaactt 540 ggtgactacg gagaagtcac actggactgt gagccaagga gtggactaaa cactgaagcg 600 ttttacgtca tgaccgtggg gtcaaagtca tttttggtcc acagggaatg gtttcatgat 660 ctcgctctcc cttggacgcc cccttcgagc acagcgtgga gaaacagaga actcctcatg 720 gaatttgaag aggcgcacgc cacaaaacag tccgttgttg ctcttgggtc acaggaagga 780 ggcctccatc aggcgttggc aggagccatc gtggtggagt actcaaactc agtgaagtta 840 acatcaggcc acctaaaatg caggctgaga atggacaaac tggctctgaa aggcacaacc 900 tatggcatgt gcacagaaaa attctcgttc gcgaaaaatc cggcggacac tggtcacgga 960 acagttgtca ttgaactttc ctactctggg agtgatggcc cttgcaaaat tccgattgtc 1020 tccgttgcga gcctcaatga catgaccccc gtcgggcggc tggtgacagt gaaccccttc 1080 gtcgcgactt ccagcgccaa ctcaaaggtg ctagtcgaga tggaaccccc cttcggagac 1140 tcctacatcg tagttggaag gggagacaag cagattaacc accattggca caaggctgga 1200 agc 1203 <210> 3 <211> 411 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence for ectodomain of Japanese encephalitis virus envelope protein (strain: Nakayama) <400> 3 Gly Ser Phe Asn Cys Leu Gly Met Gly Asn Arg Asp Phe Ile Glu Gly 1 5 10 15 Ala Ser Gly Ala Thr Trp Val Asp Leu Val Leu Glu Gly Asp Ser Cys 20 25 30 Leu Thr Ile Met Ale Asn Asp Lys Pro Thr Leu Asp Val Arg Ile 35 40 45 Asn Ile Glu Ala Val Gln Leu Ala Glu Val Arg Ser Tyr Cys Tyr His 50 55 60 Ala Ser Val Thr Asp Ile Ser Thr Val Ala Arg Cys Pro Thr Thr Gly 65 70 75 80 Glu Ala His Asn Lys Lys Arg Ala Asp Ser Ser Tyr Val Cys Lys Gln 85 90 95 Gly Phe Thr Asp Arg Gly Trp Gly Asn Gly Cys Gly Leu Phe Gly Lys 100 105 110 Gly Ser Ile Asp Thr Cys Ala Lys Phe Ser Cys Thr Ser Lys Ala Ile 115 120 125 Gly Arg Thr Ile Gln Pro Glu Asn Ile Lys Tyr Glu Val Gly Ile Phe 130 135 140 Val His Gly Thr Thr Thr Ser Glu Asn His Gly Asn Tyr Ser Ala Gln 145 150 155 160 Val Gly Ala Ser Gln Ala Ala Lys Phe Thr Val Thr Pro Asn Ala Pro 165 170 175 Ser Ile Thr Leu Lys Leu Gly Asp Tyr Gly Glu Val Thr Leu Asp Cys 180 185 190 Glu Pro Arg Ser Gly Leu Asn Thr Glu Ala Phe Tyr Val Met Thr Val 195 200 205 Gly Ser Lys Ser Phe Leu Val His Arg Glu Trp Phe His Asp Leu Ala 210 215 220 Leu Pro Trp Thr Pro Pro Ser Ser Thr Ala Trp Arg Asn Arg Glu Leu 225 230 235 240 Leu Met Glu Phe Glu Glu Ala His Ala Thr Lys Gln Ser Val Val Ala 245 250 255 Leu Gly Ser Gln Gly Gly Gly Leu His Gln Ala Leu Ala Gly Ala Ile 260 265 270 Val Val Glu Tyr Ser Asn Ser Val Lys Leu Thr Ser Gly His Leu Lys 275 280 285 Cys Arg Leu Arg Met Asp Lys Leu Ala Leu Lys Gly Thr Thr Tyr Gly 290 295 300 Met Cys Thr Glu Lys Phe Ser Phe Ala Lys Asn Pro Ala Asp Thr Gly 305 310 315 320 His Gly Thr Val Valle Glu Leu Ser Tyr Ser Gly Ser Asp Gly Pro 325 330 335 Cys Lys Ile Pro Ile Val Ser Val Ala Ser Leu Asn Asp Met Thr Pro 340 345 350 Val Gly Arg Leu Val Thr Val Asn Pro Phe Val Ala Thr Ser Ser Ala 355 360 365 Asn Ser Lys Val Leu Val Glu Met Glu Pro Pro Phe Gly Asp Ser Tyr 370 375 380 Ile Val Val Gly Arg Gly Asp Lys Gln Ile Asn His His Trp His Lys 385 390 395 400 Ala Gly Ser Leu Glu His His His His His 405 410 <210> 4 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Binding site on ectodomain of Japanese encephalitis virus envelope protein <400> 4 Phe Asn Cys Leu Gly Met Gly Asn Arg Asp 1 5 10 <210> 5 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Binding site on ectodomain of Japanese encephalitis virus envelope protein <400> 5 Val Leu Glu Gly Asp Ser Cys Leu Thr 1 5 <210> 6 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Binding site on ectodomain of Japanese encephalitis virus envelope protein <400> 6 His Ala Ser Val Thr Asp Ile Ser 1 5 <210> 7 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Binding site on ectodomain of Japanese encephalitis virus envelope protein <400> 7 Gly Trp Gly Asn Gly Cys Gly Leu 1 5 <210> 8 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Binding site on ectodomain of Japanese encephalitis virus envelope protein <400> 8 Thr Thr Ser Glu Asn His Gly Asn 1 5 <210> 9 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Binding site on ectodomain of Japanese encephalitis virus envelope protein <400> 9 Ala His Ala Thr Lys Gln Ser Val Val Ala 1 5 10 <210> 10 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Binding site on ectodomain of Japanese encephalitis virus envelope protein <400> 10 Gln Gly Gly Gly Leu His Gln Ala Leu Ala Gly Ala Ile Val Val Glu 1 5 10 15 Tyr Ser <210> 11 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Binding site on ectodomain of Japanese encephalitis virus envelope <400> 11 Asp Thr Gly His Gly Thr Val 1 5
Claims (16)
상기 돌출 도메인이 FNCLGMGNRD(서열번호 4), VLEGDSCLT(서열번호 5), HASVTDIS(서열번호 6), GWGNGCGL(서열번호 7), TTTSENHGN(서열번호 8), AHATKQSVVA(서열번호 9), QEGGLHQALAGAIVVEYS(서열번호 10), 및 DTGHGTV(서열번호 11)의 아미노산 서열을 포함하는 것인, 단백질.The method according to claim 1,
(SEQ ID NO: 4), VLEGDSCLT (SEQ ID NO: 5), HASVTDIS (SEQ ID NO: 6), GWGNGCGL (SEQ ID NO: 7), TTTSENHGN (SEQ ID NO: 8), AHATKQSVVA 10), and DTGHGTV (SEQ ID NO: 11).
상기 돌출 도메인은 서열번호 3의 아미노산 서열을 갖는 폴리펩티드 또는 이의 단편인 것을 특징으로 하는, 단백질.The method according to claim 1,
Wherein the protruding domain is a polypeptide having the amino acid sequence of SEQ ID NO: 3 or a fragment thereof.
상기 서열번호 3의 아미노산 서열을 갖는 폴리펩티드는 서열번호 2의 염기서열에 의해 코딩되는 것을 특징으로 하는, 단백질.The method of claim 3,
Wherein the polypeptide having the amino acid sequence of SEQ ID NO: 3 is encoded by the nucleotide sequence of SEQ ID NO: 2.
상기 질환은 뇌염, 무균성수막염, 열성두통 및 이들의 조합으로 구성된 군으로부터 선택되는 것을 특징으로 하는, 백신.6. The method of claim 5,
Wherein said disease is selected from the group consisting of encephalitis, aseptic meningitis, febrile headache, and combinations thereof.
상기 숙주세포는 바큘로바이러스에 의해 추가로 형질감염되는 것을 특징으로 하는, 숙주세포.12. The method of claim 11,
Wherein the host cell is further transfected with a baculovirus.
상기 숙주세포는 진핵세포인 것을 특징으로 하는, 숙주세포.12. The method of claim 11,
Wherein the host cell is a eukaryotic cell.
상기 진핵세포는 곤충세포인 것을 특징으로 하는, 숙주세포.14. The method of claim 13,
Wherein the eukaryotic cell is an insect cell.
(2) 상기 실험동물로부터 항체를 회수하는 단계를 포함하는 일본 뇌염 바이러스 항체 제조 방법.(1) introducing the homodimeric protein of claim 1 as an antigen into an experimental animal to induce an immune response; And
(2) recovering the antibody from the test animal.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020170157475A KR102039059B1 (en) | 2017-11-23 | 2017-11-23 | Protein comprising ectodomain of japanese encephalitis virus envelope proteins, homodimer thereof, and use thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020170157475A KR102039059B1 (en) | 2017-11-23 | 2017-11-23 | Protein comprising ectodomain of japanese encephalitis virus envelope proteins, homodimer thereof, and use thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| KR20190059652A true KR20190059652A (en) | 2019-05-31 |
| KR102039059B1 KR102039059B1 (en) | 2019-11-01 |
Family
ID=66657344
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| KR1020170157475A KR102039059B1 (en) | 2017-11-23 | 2017-11-23 | Protein comprising ectodomain of japanese encephalitis virus envelope proteins, homodimer thereof, and use thereof |
Country Status (1)
| Country | Link |
|---|---|
| KR (1) | KR102039059B1 (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017062174A1 (en) * | 2015-10-07 | 2017-04-13 | The University Of North Carolina At Chapel Hill | Methods and compositions for dengue virus vaccines and diagnostistics |
-
2017
- 2017-11-23 KR KR1020170157475A patent/KR102039059B1/en active IP Right Grant
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017062174A1 (en) * | 2015-10-07 | 2017-04-13 | The University Of North Carolina At Chapel Hill | Methods and compositions for dengue virus vaccines and diagnostistics |
Non-Patent Citations (3)
| Title |
|---|
| GENBANK: ACU42242.1 * |
| Min Soo Park, et al., 1999. Immune Response to SA14-14-2 Live Attenuated Japanese Encephalitis Vaccine. Department of Pediatrics, Yonsei University College of Medicine. pp. 351-358. |
| V.C. Luca 등, Journal of Virology, Vol.86, No.4, p.2337-2346(2011.12.07.)* * |
Also Published As
| Publication number | Publication date |
|---|---|
| KR102039059B1 (en) | 2019-11-01 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US11926649B2 (en) | Conformationally stabilized RSV pre-fusion F proteins | |
| EP3024483B1 (en) | Conformationally stabilized rsv pre-fusion f proteins | |
| CN105669838B (en) | Neutralizing epitopes from varicella-zoster virus gE protein and antibodies thereto | |
| WO2022184027A1 (en) | Novel coronavirus multivalent antigen, and preparation method therefor and application thereof | |
| JPH03502687A (en) | Respiratory syncytial viruses: vaccines and diagnostics | |
| KR20010052767A (en) | Vaccine | |
| KR20190035542A (en) | Nc fusion protein comprising n-terminus domain fragments and c-terminus domain fragments of mers-cov nucleocapsid protein and kit for diagnosing infection of mers-cov using the same | |
| JP2008505050A (en) | SARS coronavirus S protein and use thereof | |
| CN113354717A (en) | Novel coronavirus SARS-CoV-2 broad-spectrum polypeptide antigen and its specific neutralizing antibody and application | |
| CN116836243A (en) | F protein mutant before fusion of respiratory syncytial virus and application thereof | |
| CN105085672B (en) | 3D protein specific monoclonal immunoglobulin A antibodies and compositions thereof | |
| US11464850B2 (en) | Recombinant RSV antigens, nucleic acid molecules encoding the antigens, and vaccine compositions comprising the same | |
| KR102039059B1 (en) | Protein comprising ectodomain of japanese encephalitis virus envelope proteins, homodimer thereof, and use thereof | |
| CN111948387B (en) | Application of mycobacterium tuberculosis antigen protein Rv1485 in preparation of tuberculosis vaccine | |
| CN115322247A (en) | Novel charge mutant antigen of coronavirus receptor binding region and application | |
| WO1998014585A1 (en) | Nucleocapsid gene of seoul virus r22, recombinant plasmid, transformed e. coli and diagnostic agent and vaccine for haemorrhagic fever with renal syndrome | |
| JP2020506875A (en) | Vaccine composition comprising hepatitis B virus-like particles as adjuvant | |
| KR102006869B1 (en) | N-terminus domain fragments, c-terminus domain fragments and nc fusion protein of ebola virus nucleoprotein, kit for diagnosing ebola virus infection using thereof | |
| US11801292B2 (en) | Polypeptide epitopes of S. aureus and respective monoclonal antibodies for the treatment of infections and immune-diagnosis | |
| TWI653049B (en) | Synthetic polypeptide, vaccine composition comprising the same, and use thereof | |
| KR101991577B1 (en) | Recombinant antigenic protein composed of multiple epitopes and preparation method thereof | |
| EP4313138A1 (en) | Sars-cov-2 subunit vaccine | |
| KR20220040423A (en) | Vaccine composition for preventing or treating infection of SARS-CoV-2 comprising a recombinant protein | |
| KR20220139810A (en) | Neutralization-enhancing vaccine composition for preventing sars coronavirus 2 infection disease | |
| AU2022320680A1 (en) | Recombinant antigen for inducing an immune response against the zika virus |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A201 | Request for examination | ||
| A302 | Request for accelerated examination | ||
| E902 | Notification of reason for refusal | ||
| AMND | Amendment | ||
| E601 | Decision to refuse application | ||
| AMND | Amendment | ||
| E902 | Notification of reason for refusal | ||
| AMND | Amendment | ||
| X701 | Decision to grant (after re-examination) |