KR101763745B1 - Monoclonal antibody against envelope domain Ⅲ of four serotype dengue virus and uses thereof - Google Patents
Monoclonal antibody against envelope domain Ⅲ of four serotype dengue virus and uses thereof Download PDFInfo
- Publication number
- KR101763745B1 KR101763745B1 KR1020160002465A KR20160002465A KR101763745B1 KR 101763745 B1 KR101763745 B1 KR 101763745B1 KR 1020160002465 A KR1020160002465 A KR 1020160002465A KR 20160002465 A KR20160002465 A KR 20160002465A KR 101763745 B1 KR101763745 B1 KR 101763745B1
- Authority
- KR
- South Korea
- Prior art keywords
- monoclonal antibody
- dengue virus
- antibody
- fragment
- light chain
- Prior art date
Links
- 241000725619 Dengue virus Species 0.000 title claims abstract description 38
- 206010012310 Dengue fever Diseases 0.000 claims abstract description 37
- 208000025729 dengue disease Diseases 0.000 claims abstract description 32
- 239000012528 membrane Substances 0.000 claims abstract description 19
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 11
- 108090000623 proteins and genes Proteins 0.000 claims description 41
- 239000012634 fragment Substances 0.000 claims description 35
- 238000000034 method Methods 0.000 claims description 23
- 102000004169 proteins and genes Human genes 0.000 claims description 21
- 239000013604 expression vector Substances 0.000 claims description 15
- 239000000203 mixture Substances 0.000 claims description 15
- 108091033319 polynucleotide Proteins 0.000 claims description 13
- 102000040430 polynucleotide Human genes 0.000 claims description 13
- 239000002157 polynucleotide Substances 0.000 claims description 13
- 239000001963 growth medium Substances 0.000 claims description 8
- 238000012258 culturing Methods 0.000 claims description 7
- 239000012472 biological sample Substances 0.000 claims description 6
- 239000003937 drug carrier Substances 0.000 claims description 6
- 239000008194 pharmaceutical composition Substances 0.000 claims description 5
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims description 3
- 239000004480 active ingredient Substances 0.000 claims description 3
- 125000003275 alpha amino acid group Chemical group 0.000 claims 3
- 238000012300 Sequence Analysis Methods 0.000 abstract description 3
- 238000005516 engineering process Methods 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 38
- 108091007433 antigens Proteins 0.000 description 22
- 102000036639 antigens Human genes 0.000 description 22
- 239000000427 antigen Substances 0.000 description 21
- 235000018102 proteins Nutrition 0.000 description 18
- 239000013598 vector Substances 0.000 description 18
- 210000004408 hybridoma Anatomy 0.000 description 16
- 150000001413 amino acids Chemical group 0.000 description 13
- 238000002965 ELISA Methods 0.000 description 11
- 108010047041 Complementarity Determining Regions Proteins 0.000 description 10
- 239000000243 solution Substances 0.000 description 9
- 238000001514 detection method Methods 0.000 description 8
- 108090000790 Enzymes Proteins 0.000 description 7
- 102000004190 Enzymes Human genes 0.000 description 7
- 241000699666 Mus <mouse, genus> Species 0.000 description 7
- 241000700605 Viruses Species 0.000 description 7
- 229940088598 enzyme Drugs 0.000 description 7
- 229920001223 polyethylene glycol Polymers 0.000 description 7
- 208000001490 Dengue Diseases 0.000 description 6
- 241000710829 Dengue virus group Species 0.000 description 6
- 239000002202 Polyethylene glycol Substances 0.000 description 6
- 230000006320 pegylation Effects 0.000 description 6
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 5
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 5
- 206010057190 Respiratory tract infections Diseases 0.000 description 5
- 208000009714 Severe Dengue Diseases 0.000 description 5
- 235000001014 amino acid Nutrition 0.000 description 5
- 229940024606 amino acid Drugs 0.000 description 5
- 210000004369 blood Anatomy 0.000 description 5
- 239000008280 blood Substances 0.000 description 5
- 230000014509 gene expression Effects 0.000 description 5
- 238000004519 manufacturing process Methods 0.000 description 5
- 239000007787 solid Substances 0.000 description 5
- 210000003501 vero cell Anatomy 0.000 description 5
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 4
- 108020004414 DNA Proteins 0.000 description 4
- 101710204837 Envelope small membrane protein Proteins 0.000 description 4
- 101710145006 Lysis protein Proteins 0.000 description 4
- 241000699670 Mus sp. Species 0.000 description 4
- 101710116435 Outer membrane protein Proteins 0.000 description 4
- 206010037660 Pyrexia Diseases 0.000 description 4
- 239000013592 cell lysate Substances 0.000 description 4
- 201000009892 dengue shock syndrome Diseases 0.000 description 4
- 239000003085 diluting agent Substances 0.000 description 4
- 201000010099 disease Diseases 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 238000009472 formulation Methods 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 239000002773 nucleotide Substances 0.000 description 4
- 125000003729 nucleotide group Chemical group 0.000 description 4
- 239000000546 pharmaceutical excipient Substances 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 4
- 108091026890 Coding region Proteins 0.000 description 3
- 241000588724 Escherichia coli Species 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 208000032843 Hemorrhage Diseases 0.000 description 3
- 241000725303 Human immunodeficiency virus Species 0.000 description 3
- 108700005091 Immunoglobulin Genes Proteins 0.000 description 3
- 206010035226 Plasma cell myeloma Diseases 0.000 description 3
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 230000000740 bleeding effect Effects 0.000 description 3
- 229940098773 bovine serum albumin Drugs 0.000 description 3
- 210000000038 chest Anatomy 0.000 description 3
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 3
- 230000000295 complement effect Effects 0.000 description 3
- 239000000839 emulsion Substances 0.000 description 3
- 210000003527 eukaryotic cell Anatomy 0.000 description 3
- 239000012091 fetal bovine serum Substances 0.000 description 3
- 230000013595 glycosylation Effects 0.000 description 3
- 238000006206 glycosylation reaction Methods 0.000 description 3
- 238000003125 immunofluorescent labeling Methods 0.000 description 3
- 210000001165 lymph node Anatomy 0.000 description 3
- 201000000050 myeloid neoplasm Diseases 0.000 description 3
- 238000001556 precipitation Methods 0.000 description 3
- 230000009257 reactivity Effects 0.000 description 3
- 230000001850 reproductive effect Effects 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 230000009466 transformation Effects 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 238000001262 western blot Methods 0.000 description 3
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- 208000019901 Anxiety disease Diseases 0.000 description 2
- GGRSYTUJHAZTFN-IHRRRGAJSA-N Asp-Pro-Tyr Chemical compound C1C[C@H](N(C1)C(=O)[C@H](CC(=O)O)N)C(=O)N[C@@H](CC2=CC=C(C=C2)O)C(=O)O GGRSYTUJHAZTFN-IHRRRGAJSA-N 0.000 description 2
- HTSSXFASOUSJQG-IHPCNDPISA-N Asp-Tyr-Trp Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O HTSSXFASOUSJQG-IHPCNDPISA-N 0.000 description 2
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 2
- 241000282693 Cercopithecidae Species 0.000 description 2
- 108020004705 Codon Proteins 0.000 description 2
- ZGERHCJBLPQPGV-ACZMJKKPSA-N Cys-Ser-Gln Chemical compound C(CC(=O)N)[C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](CS)N ZGERHCJBLPQPGV-ACZMJKKPSA-N 0.000 description 2
- 241000710815 Dengue virus 2 Species 0.000 description 2
- 238000009007 Diagnostic Kit Methods 0.000 description 2
- 208000010201 Exanthema Diseases 0.000 description 2
- 241000233866 Fungi Species 0.000 description 2
- GBYYQVBXFVDJPJ-WLTAIBSBSA-N Gly-Tyr-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)NC(=O)CN)O GBYYQVBXFVDJPJ-WLTAIBSBSA-N 0.000 description 2
- 241000701044 Human gammaherpesvirus 4 Species 0.000 description 2
- 101150008942 J gene Proteins 0.000 description 2
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 2
- CLNJSLSHKJECME-BQBZGAKWSA-N Pro-Gly-Ala Chemical compound OC(=O)[C@H](C)NC(=O)CNC(=O)[C@@H]1CCCN1 CLNJSLSHKJECME-BQBZGAKWSA-N 0.000 description 2
- 108010076504 Protein Sorting Signals Proteins 0.000 description 2
- 241000235070 Saccharomyces Species 0.000 description 2
- QNBVFKZSSRYNFX-CUJWVEQBSA-N Ser-Thr-His Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)NC(=O)[C@H](CO)N)O QNBVFKZSSRYNFX-CUJWVEQBSA-N 0.000 description 2
- 241001147693 Staphylococcus sp. Species 0.000 description 2
- 108091081024 Start codon Proteins 0.000 description 2
- 108020005038 Terminator Codon Proteins 0.000 description 2
- HUPLKEHTTQBXSC-YJRXYDGGSA-N Thr-Ser-Tyr Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 HUPLKEHTTQBXSC-YJRXYDGGSA-N 0.000 description 2
- 101710120037 Toxin CcdB Proteins 0.000 description 2
- 101150117115 V gene Proteins 0.000 description 2
- HQYVQDRYODWONX-DCAQKATOSA-N Val-His-Ser Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)N[C@@H](CO)C(=O)O)N HQYVQDRYODWONX-DCAQKATOSA-N 0.000 description 2
- 210000001015 abdomen Anatomy 0.000 description 2
- 238000001042 affinity chromatography Methods 0.000 description 2
- 108010087924 alanylproline Proteins 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000036506 anxiety Effects 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 239000011230 binding agent Substances 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
- 239000002775 capsule Substances 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- CVSVTCORWBXHQV-UHFFFAOYSA-N creatine Chemical compound NC(=[NH2+])N(C)CC([O-])=O CVSVTCORWBXHQV-UHFFFAOYSA-N 0.000 description 2
- 239000013078 crystal Substances 0.000 description 2
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 201000005884 exanthem Diseases 0.000 description 2
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 230000004927 fusion Effects 0.000 description 2
- 125000003147 glycosyl group Chemical group 0.000 description 2
- 239000008187 granular material Substances 0.000 description 2
- 230000002163 immunogen Effects 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 239000008101 lactose Substances 0.000 description 2
- 239000003446 ligand Substances 0.000 description 2
- 239000000314 lubricant Substances 0.000 description 2
- HQKMJHAJHXVSDF-UHFFFAOYSA-L magnesium stearate Chemical compound [Mg+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O HQKMJHAJHXVSDF-UHFFFAOYSA-L 0.000 description 2
- 230000014759 maintenance of location Effects 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 239000002105 nanoparticle Substances 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 239000006187 pill Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000003672 processing method Methods 0.000 description 2
- 210000001236 prokaryotic cell Anatomy 0.000 description 2
- 108010029020 prolylglycine Proteins 0.000 description 2
- 230000002285 radioactive effect Effects 0.000 description 2
- 206010037844 rash Diseases 0.000 description 2
- 238000003118 sandwich ELISA Methods 0.000 description 2
- 210000004989 spleen cell Anatomy 0.000 description 2
- 239000004094 surface-active agent Substances 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 239000003826 tablet Substances 0.000 description 2
- 239000000080 wetting agent Substances 0.000 description 2
- DIGQNXIGRZPYDK-WKSCXVIASA-N (2R)-6-amino-2-[[2-[[(2S)-2-[[2-[[(2R)-2-[[(2S)-2-[[(2R,3S)-2-[[2-[[(2S)-2-[[2-[[(2S)-2-[[(2S)-2-[[(2R)-2-[[(2S,3S)-2-[[(2R)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[2-[[(2S)-2-[[(2R)-2-[[2-[[2-[[2-[(2-amino-1-hydroxyethylidene)amino]-3-carboxy-1-hydroxypropylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1-hydroxyethylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxyethylidene]amino]-1-hydroxypropylidene]amino]-1,3-dihydroxypropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxybutylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1-hydroxypropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxyethylidene]amino]-1,5-dihydroxy-5-iminopentylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxybutylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxyethylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1-hydroxyethylidene]amino]hexanoic acid Chemical compound C[C@@H]([C@@H](C(=N[C@@H](CS)C(=N[C@@H](C)C(=N[C@@H](CO)C(=NCC(=N[C@@H](CCC(=N)O)C(=NC(CS)C(=N[C@H]([C@H](C)O)C(=N[C@H](CS)C(=N[C@H](CO)C(=NCC(=N[C@H](CS)C(=NCC(=N[C@H](CCCCN)C(=O)O)O)O)O)O)O)O)O)O)O)O)O)O)O)N=C([C@H](CS)N=C([C@H](CO)N=C([C@H](CO)N=C([C@H](C)N=C(CN=C([C@H](CO)N=C([C@H](CS)N=C(CN=C(C(CS)N=C(C(CC(=O)O)N=C(CN)O)O)O)O)O)O)O)O)O)O)O)O DIGQNXIGRZPYDK-WKSCXVIASA-N 0.000 description 1
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 description 1
- UHEPSJJJMTWUCP-DHDYTCSHSA-N (2r,3r,4r,5r)-2-[(1s,2s,3r,4s,6r)-4,6-diamino-3-[(2s,3r,4r,5s,6r)-3-amino-4,5-dihydroxy-6-[(1r)-1-hydroxyethyl]oxan-2-yl]oxy-2-hydroxycyclohexyl]oxy-5-methyl-4-(methylamino)oxane-3,5-diol;sulfuric acid Chemical compound OS(O)(=O)=O.OS(O)(=O)=O.O1C[C@@](O)(C)[C@H](NC)[C@@H](O)[C@H]1O[C@@H]1[C@@H](O)[C@H](O[C@@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H]([C@@H](C)O)O2)N)[C@@H](N)C[C@H]1N UHEPSJJJMTWUCP-DHDYTCSHSA-N 0.000 description 1
- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 description 1
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 1
- XZKIHKMTEMTJQX-UHFFFAOYSA-N 4-Nitrophenyl Phosphate Chemical compound OP(O)(=O)OC1=CC=C([N+]([O-])=O)C=C1 XZKIHKMTEMTJQX-UHFFFAOYSA-N 0.000 description 1
- HUDPLKWXRLNSPC-UHFFFAOYSA-N 4-aminophthalhydrazide Chemical class O=C1NNC(=O)C=2C1=CC(N)=CC=2 HUDPLKWXRLNSPC-UHFFFAOYSA-N 0.000 description 1
- 102000007469 Actins Human genes 0.000 description 1
- 108010085238 Actins Proteins 0.000 description 1
- 241000589158 Agrobacterium Species 0.000 description 1
- DWINFPQUSSHSFS-UVBJJODRSA-N Ala-Arg-Trp Chemical compound N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC1=CNC2=CC=CC=C12)C(=O)O DWINFPQUSSHSFS-UVBJJODRSA-N 0.000 description 1
- OINVDEKBKBCPLX-JXUBOQSCSA-N Ala-Lys-Thr Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(O)=O OINVDEKBKBCPLX-JXUBOQSCSA-N 0.000 description 1
- DYXOFPBJBAHWFY-JBDRJPRFSA-N Ala-Ser-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@H](C)N DYXOFPBJBAHWFY-JBDRJPRFSA-N 0.000 description 1
- NCQMBSJGJMYKCK-ZLUOBGJFSA-N Ala-Ser-Ser Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O NCQMBSJGJMYKCK-ZLUOBGJFSA-N 0.000 description 1
- WNHNMKOFKCHKKD-BFHQHQDPSA-N Ala-Thr-Gly Chemical compound [H]N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(O)=O WNHNMKOFKCHKKD-BFHQHQDPSA-N 0.000 description 1
- IOFVWPYSRSCWHI-JXUBOQSCSA-N Ala-Thr-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](C)N IOFVWPYSRSCWHI-JXUBOQSCSA-N 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- FRBAHXABMQXSJQ-FXQIFTODSA-N Arg-Ser-Ser Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O FRBAHXABMQXSJQ-FXQIFTODSA-N 0.000 description 1
- 240000003291 Armoracia rusticana Species 0.000 description 1
- 235000011330 Armoracia rusticana Nutrition 0.000 description 1
- 208000006820 Arthralgia Diseases 0.000 description 1
- DXVMJJNAOVECBA-WHFBIAKZSA-N Asn-Gly-Asn Chemical compound NC(=O)C[C@H](N)C(=O)NCC(=O)N[C@@H](CC(N)=O)C(O)=O DXVMJJNAOVECBA-WHFBIAKZSA-N 0.000 description 1
- UDSVWSUXKYXSTR-QWRGUYRKSA-N Asn-Gly-Tyr Chemical compound [H]N[C@@H](CC(N)=O)C(=O)NCC(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O UDSVWSUXKYXSTR-QWRGUYRKSA-N 0.000 description 1
- VOKWBBBXJONREA-DCAQKATOSA-N Asn-Met-His Chemical compound CSCC[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)NC(=O)[C@H](CC(=O)N)N VOKWBBBXJONREA-DCAQKATOSA-N 0.000 description 1
- KZYSHAMXEBPJBD-JRQIVUDYSA-N Asn-Thr-Tyr Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O KZYSHAMXEBPJBD-JRQIVUDYSA-N 0.000 description 1
- JPPLRQVZMZFOSX-UWJYBYFXSA-N Asn-Tyr-Ala Chemical compound NC(=O)C[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](C)C(O)=O)CC1=CC=C(O)C=C1 JPPLRQVZMZFOSX-UWJYBYFXSA-N 0.000 description 1
- KRXIWXCXOARFNT-ZLUOBGJFSA-N Asp-Ala-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC(O)=O KRXIWXCXOARFNT-ZLUOBGJFSA-N 0.000 description 1
- WMLFFCRUSPNENW-ZLUOBGJFSA-N Asp-Ser-Ala Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(O)=O WMLFFCRUSPNENW-ZLUOBGJFSA-N 0.000 description 1
- ZUNMTUPRQMWMHX-LSJOCFKGSA-N Asp-Val-Val Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C(C)C)C(O)=O ZUNMTUPRQMWMHX-LSJOCFKGSA-N 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Chemical group OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 1
- 241000228257 Aspergillus sp. Species 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 241000699800 Cricetinae Species 0.000 description 1
- AMRLSQGGERHDHJ-FXQIFTODSA-N Cys-Ala-Arg Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O AMRLSQGGERHDHJ-FXQIFTODSA-N 0.000 description 1
- 101150097493 D gene Proteins 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 230000007018 DNA scission Effects 0.000 description 1
- 241000710827 Dengue virus 1 Species 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- LVGKNOAMLMIIKO-UHFFFAOYSA-N Elaidinsaeure-aethylester Natural products CCCCCCCCC=CCCCCCCCC(=O)OCC LVGKNOAMLMIIKO-UHFFFAOYSA-N 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 101710091045 Envelope protein Proteins 0.000 description 1
- 108090000371 Esterases Proteins 0.000 description 1
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 208000015220 Febrile disease Diseases 0.000 description 1
- 239000004606 Fillers/Extenders Substances 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- JXFLPKSDLDEOQK-JHEQGTHGSA-N Gln-Gly-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CCC(N)=O JXFLPKSDLDEOQK-JHEQGTHGSA-N 0.000 description 1
- ILKYYKRAULNYMS-JYJNAYRXSA-N Gln-Lys-Phe Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O ILKYYKRAULNYMS-JYJNAYRXSA-N 0.000 description 1
- LPIKVBWNNVFHCQ-GUBZILKMSA-N Gln-Ser-Leu Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(O)=O LPIKVBWNNVFHCQ-GUBZILKMSA-N 0.000 description 1
- VLOLPWWCNKWRNB-LOKLDPHHSA-N Gln-Thr-Pro Chemical compound C[C@H]([C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CCC(=O)N)N)O VLOLPWWCNKWRNB-LOKLDPHHSA-N 0.000 description 1
- ZFBBMCKQSNJZSN-AUTRQRHGSA-N Gln-Val-Gln Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O ZFBBMCKQSNJZSN-AUTRQRHGSA-N 0.000 description 1
- JVSBYEDSSRZQGV-GUBZILKMSA-N Glu-Asp-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](N)CCC(O)=O JVSBYEDSSRZQGV-GUBZILKMSA-N 0.000 description 1
- GJBUAAAIZSRCDC-GVXVVHGQSA-N Glu-Leu-Val Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(O)=O GJBUAAAIZSRCDC-GVXVVHGQSA-N 0.000 description 1
- JVZLZVJTIXVIHK-SXNHZJKMSA-N Glu-Trp-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC1=CNC2=CC=CC=C21)NC(=O)[C@H](CCC(=O)O)N JVZLZVJTIXVIHK-SXNHZJKMSA-N 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- YMUFWNJHVPQNQD-ZKWXMUAHSA-N Gly-Ala-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)CN YMUFWNJHVPQNQD-ZKWXMUAHSA-N 0.000 description 1
- GGEJHJIXRBTJPD-BYPYZUCNSA-N Gly-Asn-Gly Chemical compound NCC(=O)N[C@@H](CC(N)=O)C(=O)NCC(O)=O GGEJHJIXRBTJPD-BYPYZUCNSA-N 0.000 description 1
- XEJTYSCIXKYSHR-WDSKDSINSA-N Gly-Asp-Gln Chemical compound C(CC(=O)N)[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)CN XEJTYSCIXKYSHR-WDSKDSINSA-N 0.000 description 1
- BYYNJRSNDARRBX-YFKPBYRVSA-N Gly-Gln-Gly Chemical compound NCC(=O)N[C@@H](CCC(N)=O)C(=O)NCC(O)=O BYYNJRSNDARRBX-YFKPBYRVSA-N 0.000 description 1
- UQJNXZSSGQIPIQ-FBCQKBJTSA-N Gly-Gly-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)CNC(=O)CN UQJNXZSSGQIPIQ-FBCQKBJTSA-N 0.000 description 1
- NVTPVQLIZCOJFK-FOHZUACHSA-N Gly-Thr-Asp Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(O)=O)C(O)=O NVTPVQLIZCOJFK-FOHZUACHSA-N 0.000 description 1
- IZVICCORZOSGPT-JSGCOSHPSA-N Gly-Val-Tyr Chemical compound [H]NCC(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O IZVICCORZOSGPT-JSGCOSHPSA-N 0.000 description 1
- 102000051366 Glycosyltransferases Human genes 0.000 description 1
- 108700023372 Glycosyltransferases Proteins 0.000 description 1
- JBCLFWXMTIKCCB-UHFFFAOYSA-N H-Gly-Phe-OH Natural products NCC(=O)NC(C(O)=O)CC1=CC=CC=C1 JBCLFWXMTIKCCB-UHFFFAOYSA-N 0.000 description 1
- 102000001554 Hemoglobins Human genes 0.000 description 1
- 108010054147 Hemoglobins Proteins 0.000 description 1
- 229920002971 Heparan sulfate Polymers 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101000756632 Homo sapiens Actin, cytoplasmic 1 Proteins 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- IOVUXUSIGXCREV-DKIMLUQUSA-N Ile-Leu-Phe Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 IOVUXUSIGXCREV-DKIMLUQUSA-N 0.000 description 1
- JHNJNTMTZHEDLJ-NAKRPEOUSA-N Ile-Ser-Arg Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O JHNJNTMTZHEDLJ-NAKRPEOUSA-N 0.000 description 1
- NSPNUMNLZNOPAQ-SJWGOKEGSA-N Ile-Tyr-Pro Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N2CCC[C@@H]2C(=O)O)N NSPNUMNLZNOPAQ-SJWGOKEGSA-N 0.000 description 1
- 206010021432 Immunisation reaction Diseases 0.000 description 1
- 108700029228 Immunoglobulin Heavy Chain Genes Proteins 0.000 description 1
- 102000013463 Immunoglobulin Light Chains Human genes 0.000 description 1
- 108010065825 Immunoglobulin Light Chains Proteins 0.000 description 1
- 108010067060 Immunoglobulin Variable Region Proteins 0.000 description 1
- 102000017727 Immunoglobulin Variable Region Human genes 0.000 description 1
- 102100034349 Integrase Human genes 0.000 description 1
- 241000235058 Komagataella pastoris Species 0.000 description 1
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical group OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 1
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical group C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 1
- TYYLDKGBCJGJGW-UHFFFAOYSA-N L-tryptophan-L-tyrosine Natural products C=1NC2=CC=CC=C2C=1CC(N)C(=O)NC(C(O)=O)CC1=CC=C(O)C=C1 TYYLDKGBCJGJGW-UHFFFAOYSA-N 0.000 description 1
- HBJZFCIVFIBNSV-DCAQKATOSA-N Leu-Arg-Asn Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CC(N)=O)C(O)=O HBJZFCIVFIBNSV-DCAQKATOSA-N 0.000 description 1
- KAFOIVJDVSZUMD-DCAQKATOSA-N Leu-Gln-Gln Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O KAFOIVJDVSZUMD-DCAQKATOSA-N 0.000 description 1
- KAFOIVJDVSZUMD-UHFFFAOYSA-N Leu-Gln-Gln Natural products CC(C)CC(N)C(=O)NC(CCC(N)=O)C(=O)NC(CCC(N)=O)C(O)=O KAFOIVJDVSZUMD-UHFFFAOYSA-N 0.000 description 1
- HPBCTWSUJOGJSH-MNXVOIDGSA-N Leu-Glu-Ile Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O HPBCTWSUJOGJSH-MNXVOIDGSA-N 0.000 description 1
- LKXANTUNFMVCNF-IHPCNDPISA-N Leu-His-Trp Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O LKXANTUNFMVCNF-IHPCNDPISA-N 0.000 description 1
- FAELBUXXFQLUAX-AJNGGQMLSA-N Leu-Leu-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CC(C)C FAELBUXXFQLUAX-AJNGGQMLSA-N 0.000 description 1
- LFXSPAIBSZSTEM-PMVMPFDFSA-N Leu-Trp-Phe Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)N[C@@H](CC3=CC=CC=C3)C(=O)O)N LFXSPAIBSZSTEM-PMVMPFDFSA-N 0.000 description 1
- LMDVGHQPPPLYAR-IHRRRGAJSA-N Leu-Val-His Chemical compound N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC1=CNC=N1)C(=O)O LMDVGHQPPPLYAR-IHRRRGAJSA-N 0.000 description 1
- UWKNTTJNVSYXPC-CIUDSAMLSA-N Lys-Ala-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CCCCN UWKNTTJNVSYXPC-CIUDSAMLSA-N 0.000 description 1
- WXJKFRMKJORORD-DCAQKATOSA-N Lys-Arg-Ala Chemical compound NC(=N)NCCC[C@@H](C(=O)N[C@@H](C)C(O)=O)NC(=O)[C@@H](N)CCCCN WXJKFRMKJORORD-DCAQKATOSA-N 0.000 description 1
- NKKFVJRLCCUJNA-QWRGUYRKSA-N Lys-Gly-Lys Chemical compound NCCCC[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCCCN NKKFVJRLCCUJNA-QWRGUYRKSA-N 0.000 description 1
- 239000005913 Maltodextrin Substances 0.000 description 1
- 229920002774 Maltodextrin Polymers 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 102000018697 Membrane Proteins Human genes 0.000 description 1
- DLAFCQWUMFMZSN-GUBZILKMSA-N Met-Arg-Ala Chemical compound CSCC[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](C)C(O)=O)CCCN=C(N)N DLAFCQWUMFMZSN-GUBZILKMSA-N 0.000 description 1
- VOOINLQYUZOREH-SRVKXCTJSA-N Met-Gln-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CCSC)N VOOINLQYUZOREH-SRVKXCTJSA-N 0.000 description 1
- MHQXIBRPDKXDGZ-ZFWWWQNUSA-N Met-Gly-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)CNC(=O)[C@@H](N)CCSC)C(O)=O)=CNC2=C1 MHQXIBRPDKXDGZ-ZFWWWQNUSA-N 0.000 description 1
- WRXOPYNEKGZWAZ-FXQIFTODSA-N Met-Ser-Cys Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CS)C(O)=O WRXOPYNEKGZWAZ-FXQIFTODSA-N 0.000 description 1
- SPSSJSICDYYTQN-HJGDQZAQSA-N Met-Thr-Gln Chemical compound CSCC[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@H](C(O)=O)CCC(N)=O SPSSJSICDYYTQN-HJGDQZAQSA-N 0.000 description 1
- 102000003792 Metallothionein Human genes 0.000 description 1
- 108090000157 Metallothionein Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000713333 Mouse mammary tumor virus Species 0.000 description 1
- 208000000112 Myalgia Diseases 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 241000221961 Neurospora crassa Species 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
- 108700026244 Open Reading Frames Proteins 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 239000002033 PVDF binder Substances 0.000 description 1
- 108090000526 Papain Proteins 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 102000057297 Pepsin A Human genes 0.000 description 1
- 108090000284 Pepsin A Proteins 0.000 description 1
- YTILBRIUASDGBL-BZSNNMDCSA-N Phe-Leu-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CC1=CC=CC=C1 YTILBRIUASDGBL-BZSNNMDCSA-N 0.000 description 1
- IWZRODDWOSIXPZ-IRXDYDNUSA-N Phe-Phe-Gly Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)NCC(O)=O)C1=CC=CC=C1 IWZRODDWOSIXPZ-IRXDYDNUSA-N 0.000 description 1
- BPCLGWHVPVTTFM-QWRGUYRKSA-N Phe-Ser-Gly Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CO)C(=O)NCC(O)=O BPCLGWHVPVTTFM-QWRGUYRKSA-N 0.000 description 1
- GNRMAQSIROFNMI-IXOXFDKPSA-N Phe-Thr-Ser Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(O)=O GNRMAQSIROFNMI-IXOXFDKPSA-N 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 229920002535 Polyethylene Glycol 1500 Polymers 0.000 description 1
- AJLVKXCNXIJHDV-CIUDSAMLSA-N Pro-Ala-Gln Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(N)=O)C(O)=O AJLVKXCNXIJHDV-CIUDSAMLSA-N 0.000 description 1
- JARJPEMLQAWNBR-GUBZILKMSA-N Pro-Asp-Arg Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O JARJPEMLQAWNBR-GUBZILKMSA-N 0.000 description 1
- JMVQDLDPDBXAAX-YUMQZZPRSA-N Pro-Gly-Gln Chemical compound NC(=O)CC[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H]1CCCN1 JMVQDLDPDBXAAX-YUMQZZPRSA-N 0.000 description 1
- CXGLFEOYCJFKPR-RCWTZXSCSA-N Pro-Thr-Val Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(O)=O CXGLFEOYCJFKPR-RCWTZXSCSA-N 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 101710188315 Protein X Proteins 0.000 description 1
- 241000588770 Proteus mirabilis Species 0.000 description 1
- 241000589774 Pseudomonas sp. Species 0.000 description 1
- 238000011530 RNeasy Mini Kit Methods 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 108020005091 Replication Origin Proteins 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 101100221606 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) COS7 gene Proteins 0.000 description 1
- 241000720795 Schizosaccharomyces sp. Species 0.000 description 1
- OBXVZEAMXFSGPU-FXQIFTODSA-N Ser-Asn-Arg Chemical compound C(C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CO)N)CN=C(N)N OBXVZEAMXFSGPU-FXQIFTODSA-N 0.000 description 1
- VAUMZJHYZQXZBQ-WHFBIAKZSA-N Ser-Asn-Gly Chemical compound OC[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)NCC(O)=O VAUMZJHYZQXZBQ-WHFBIAKZSA-N 0.000 description 1
- BLPYXIXXCFVIIF-FXQIFTODSA-N Ser-Cys-Arg Chemical compound C(C[C@@H](C(=O)O)NC(=O)[C@H](CS)NC(=O)[C@H](CO)N)CN=C(N)N BLPYXIXXCFVIIF-FXQIFTODSA-N 0.000 description 1
- UIGMAMGZOJVTDN-WHFBIAKZSA-N Ser-Gly-Ser Chemical compound OC[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O UIGMAMGZOJVTDN-WHFBIAKZSA-N 0.000 description 1
- XXXAXOWMBOKTRN-XPUUQOCRSA-N Ser-Gly-Val Chemical compound [H]N[C@@H](CO)C(=O)NCC(=O)N[C@@H](C(C)C)C(O)=O XXXAXOWMBOKTRN-XPUUQOCRSA-N 0.000 description 1
- KCGIREHVWRXNDH-GARJFASQSA-N Ser-Leu-Pro Chemical compound CC(C)C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CO)N KCGIREHVWRXNDH-GARJFASQSA-N 0.000 description 1
- NMZXJDSKEGFDLJ-DCAQKATOSA-N Ser-Pro-Lys Chemical compound C1C[C@H](N(C1)C(=O)[C@H](CO)N)C(=O)N[C@@H](CCCCN)C(=O)O NMZXJDSKEGFDLJ-DCAQKATOSA-N 0.000 description 1
- FZXOPYUEQGDGMS-ACZMJKKPSA-N Ser-Ser-Gln Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(O)=O FZXOPYUEQGDGMS-ACZMJKKPSA-N 0.000 description 1
- BMKNXTJLHFIAAH-CIUDSAMLSA-N Ser-Ser-Leu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(O)=O BMKNXTJLHFIAAH-CIUDSAMLSA-N 0.000 description 1
- UBTNVMGPMYDYIU-HJPIBITLSA-N Ser-Tyr-Ile Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O UBTNVMGPMYDYIU-HJPIBITLSA-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
- SIEBDTCABMZCLF-XGEHTFHBSA-N Ser-Val-Thr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O SIEBDTCABMZCLF-XGEHTFHBSA-N 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 241000187180 Streptomyces sp. Species 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 101150006914 TRP1 gene Proteins 0.000 description 1
- 244000299461 Theobroma cacao Species 0.000 description 1
- 235000005764 Theobroma cacao ssp. cacao Nutrition 0.000 description 1
- 235000005767 Theobroma cacao ssp. sphaerocarpum Nutrition 0.000 description 1
- NJEMRSFGDNECGF-GCJQMDKQSA-N Thr-Ala-Asp Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CC(O)=O NJEMRSFGDNECGF-GCJQMDKQSA-N 0.000 description 1
- DGDCHPCRMWEOJR-FQPOAREZSA-N Thr-Ala-Tyr Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 DGDCHPCRMWEOJR-FQPOAREZSA-N 0.000 description 1
- MECLEFZMPPOEAC-VOAKCMCISA-N Thr-Leu-Lys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)O)N)O MECLEFZMPPOEAC-VOAKCMCISA-N 0.000 description 1
- BIBYEFRASCNLAA-CDMKHQONSA-N Thr-Phe-Gly Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@H](C(=O)NCC(O)=O)CC1=CC=CC=C1 BIBYEFRASCNLAA-CDMKHQONSA-N 0.000 description 1
- DEGCBBCMYWNJNA-RHYQMDGZSA-N Thr-Pro-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)[C@@H](C)O DEGCBBCMYWNJNA-RHYQMDGZSA-N 0.000 description 1
- XHWCDRUPDNSDAZ-XKBZYTNZSA-N Thr-Ser-Glu Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N)O XHWCDRUPDNSDAZ-XKBZYTNZSA-N 0.000 description 1
- JAWUQFCGNVEDRN-MEYUZBJRSA-N Thr-Tyr-Leu Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CC(C)C)C(=O)O)N)O JAWUQFCGNVEDRN-MEYUZBJRSA-N 0.000 description 1
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Chemical group CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 1
- 239000004473 Threonine Chemical group 0.000 description 1
- AIISTODACBDQLW-WDSOQIARSA-N Trp-Leu-Arg Chemical compound C1=CC=C2C(C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O)=CNC2=C1 AIISTODACBDQLW-WDSOQIARSA-N 0.000 description 1
- UOXPLPBMEPLZBW-WDSOQIARSA-N Trp-Val-Lys Chemical compound C1=CC=C2C(C[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCCN)C(O)=O)=CNC2=C1 UOXPLPBMEPLZBW-WDSOQIARSA-N 0.000 description 1
- CDRYEAWHKJSGAF-BPNCWPANSA-N Tyr-Ala-Met Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](C)C(=O)N[C@@H](CCSC)C(O)=O CDRYEAWHKJSGAF-BPNCWPANSA-N 0.000 description 1
- NKUGCYDFQKFVOJ-JYJNAYRXSA-N Tyr-Leu-Gln Chemical compound NC(=O)CC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 NKUGCYDFQKFVOJ-JYJNAYRXSA-N 0.000 description 1
- PMHLLBKTDHQMCY-ULQDDVLXSA-N Tyr-Lys-Val Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(O)=O PMHLLBKTDHQMCY-ULQDDVLXSA-N 0.000 description 1
- UPODKYBYUBTWSV-BZSNNMDCSA-N Tyr-Phe-Cys Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CS)C(O)=O)C1=CC=C(O)C=C1 UPODKYBYUBTWSV-BZSNNMDCSA-N 0.000 description 1
- SZEIFUXUTBBQFQ-STQMWFEESA-N Tyr-Pro-Gly Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N1CCC[C@H]1C(=O)NCC(O)=O SZEIFUXUTBBQFQ-STQMWFEESA-N 0.000 description 1
- WQOHKVRQDLNDIL-YJRXYDGGSA-N Tyr-Thr-Ser Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(O)=O WQOHKVRQDLNDIL-YJRXYDGGSA-N 0.000 description 1
- SLLKXDSRVAOREO-KZVJFYERSA-N Val-Ala-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](C)NC(=O)[C@H](C(C)C)N)O SLLKXDSRVAOREO-KZVJFYERSA-N 0.000 description 1
- XGJLNBNZNMVJRS-NRPADANISA-N Val-Glu-Ala Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(O)=O XGJLNBNZNMVJRS-NRPADANISA-N 0.000 description 1
- KSFXWENSJABBFI-ZKWXMUAHSA-N Val-Ser-Asn Chemical compound [H]N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(O)=O KSFXWENSJABBFI-ZKWXMUAHSA-N 0.000 description 1
- VHIZXDZMTDVFGX-DCAQKATOSA-N Val-Ser-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](C(C)C)N VHIZXDZMTDVFGX-DCAQKATOSA-N 0.000 description 1
- PZTZYZUTCPZWJH-FXQIFTODSA-N Val-Ser-Ser Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)O)N PZTZYZUTCPZWJH-FXQIFTODSA-N 0.000 description 1
- 208000011312 Vector Borne disease Diseases 0.000 description 1
- 241000212749 Zesius chrysomallus Species 0.000 description 1
- 108010081404 acein-2 Proteins 0.000 description 1
- OIPILFWXSMYKGL-UHFFFAOYSA-N acetylcholine Chemical compound CC(=O)OCC[N+](C)(C)C OIPILFWXSMYKGL-UHFFFAOYSA-N 0.000 description 1
- 229960004373 acetylcholine Drugs 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 238000013019 agitation Methods 0.000 description 1
- 108010086434 alanyl-seryl-glycine Proteins 0.000 description 1
- 108010041407 alanylaspartic acid Proteins 0.000 description 1
- 108010005233 alanylglutamic acid Proteins 0.000 description 1
- 125000003368 amide group Chemical group 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 208000022531 anorexia Diseases 0.000 description 1
- 210000000628 antibody-producing cell Anatomy 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 239000003125 aqueous solvent Substances 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- 108010069205 aspartyl-phenylalanine Proteins 0.000 description 1
- 238000011888 autopsy Methods 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 239000000022 bacteriostatic agent Substances 0.000 description 1
- 125000004057 biotinyl group Chemical class [H]N1C(=O)N([H])[C@]2([H])[C@@]([H])(SC([H])([H])[C@]12[H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C(*)=O 0.000 description 1
- 208000034158 bleeding Diseases 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000007975 buffered saline Substances 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 238000010804 cDNA synthesis Methods 0.000 description 1
- 235000001046 cacaotero Nutrition 0.000 description 1
- 229910000019 calcium carbonate Inorganic materials 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000007910 cell fusion Effects 0.000 description 1
- 230000005859 cell recognition Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000013522 chelant Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 239000000084 colloidal system Substances 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 229960003624 creatine Drugs 0.000 description 1
- 239000006046 creatine Substances 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 229940127089 cytotoxic agent Drugs 0.000 description 1
- 239000002254 cytotoxic agent Substances 0.000 description 1
- 231100000599 cytotoxic agent Toxicity 0.000 description 1
- 206010061428 decreased appetite Diseases 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 201000002950 dengue hemorrhagic fever Diseases 0.000 description 1
- 230000002542 deteriorative effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 229960000633 dextran sulfate Drugs 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- 239000007884 disintegrant Substances 0.000 description 1
- 239000002270 dispersing agent Substances 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000002848 electrochemical method Methods 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 208000001780 epistaxis Diseases 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- LVGKNOAMLMIIKO-QXMHVHEDSA-N ethyl oleate Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OCC LVGKNOAMLMIIKO-QXMHVHEDSA-N 0.000 description 1
- 229940093471 ethyl oleate Drugs 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 239000010419 fine particle Substances 0.000 description 1
- 235000003599 food sweetener Nutrition 0.000 description 1
- 239000003205 fragrance Substances 0.000 description 1
- 239000012737 fresh medium Substances 0.000 description 1
- 108010063718 gamma-glutamylaspartic acid Proteins 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 230000008571 general function Effects 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 210000004602 germ cell Anatomy 0.000 description 1
- 108010057083 glutamyl-aspartyl-leucine Proteins 0.000 description 1
- 108091005996 glycated proteins Proteins 0.000 description 1
- 230000036252 glycation Effects 0.000 description 1
- XBGGUPMXALFZOT-UHFFFAOYSA-N glycyl-L-tyrosine hemihydrate Natural products NCC(=O)NC(C(O)=O)CC1=CC=C(O)C=C1 XBGGUPMXALFZOT-UHFFFAOYSA-N 0.000 description 1
- 108010090037 glycyl-alanyl-isoleucine Proteins 0.000 description 1
- 108010019832 glycyl-asparaginyl-glycine Proteins 0.000 description 1
- XKUKSGPZAADMRA-UHFFFAOYSA-N glycyl-glycyl-glycine Natural products NCC(=O)NCC(=O)NCC(O)=O XKUKSGPZAADMRA-UHFFFAOYSA-N 0.000 description 1
- 108010050848 glycylleucine Proteins 0.000 description 1
- 108010087823 glycyltyrosine Proteins 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 208000021760 high fever Diseases 0.000 description 1
- 108010018006 histidylserine Proteins 0.000 description 1
- 229920002674 hyaluronan Polymers 0.000 description 1
- 229960003160 hyaluronic acid Drugs 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 150000004754 hydrosilicons Chemical group 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 230000036737 immune function Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 238000003119 immunoblot Methods 0.000 description 1
- 238000012760 immunocytochemical staining Methods 0.000 description 1
- 238000011532 immunohistochemical staining Methods 0.000 description 1
- 239000003547 immunosorbent Substances 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 210000003292 kidney cell Anatomy 0.000 description 1
- 239000004816 latex Substances 0.000 description 1
- 229920000126 latex Polymers 0.000 description 1
- VMPHSYLJUKZBJJ-UHFFFAOYSA-N lauric acid triglyceride Natural products CCCCCCCCCCCC(=O)OCC(OC(=O)CCCCCCCCCCC)COC(=O)CCCCCCCCCCC VMPHSYLJUKZBJJ-UHFFFAOYSA-N 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 229940057995 liquid paraffin Drugs 0.000 description 1
- 239000006193 liquid solution Substances 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 108010057952 lysyl-phenylalanyl-lysine Proteins 0.000 description 1
- 229960003511 macrogol Drugs 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 235000019359 magnesium stearate Nutrition 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 229940035034 maltodextrin Drugs 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 230000002175 menstrual effect Effects 0.000 description 1
- 239000011859 microparticle Substances 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 201000009240 nasopharyngitis Diseases 0.000 description 1
- 239000012457 nonaqueous media Substances 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- -1 olive oil Chemical compound 0.000 description 1
- 235000008390 olive oil Nutrition 0.000 description 1
- 239000004006 olive oil Substances 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 238000007500 overflow downdraw method Methods 0.000 description 1
- 229940055729 papain Drugs 0.000 description 1
- 235000019834 papain Nutrition 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 238000007911 parenteral administration Methods 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000007918 pathogenicity Effects 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- 229940111202 pepsin Drugs 0.000 description 1
- RXNXLAHQOVLMIE-UHFFFAOYSA-N phenyl 10-methylacridin-10-ium-9-carboxylate Chemical compound C12=CC=CC=C2[N+](C)=C2C=CC=CC2=C1C(=O)OC1=CC=CC=C1 RXNXLAHQOVLMIE-UHFFFAOYSA-N 0.000 description 1
- 108010070409 phenylalanyl-glycyl-glycine Proteins 0.000 description 1
- 108010084525 phenylalanyl-phenylalanyl-glycine Proteins 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 210000002381 plasma Anatomy 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 230000008488 polyadenylation Effects 0.000 description 1
- 229920002704 polyhistidine Polymers 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 108020001580 protein domains Proteins 0.000 description 1
- 210000001938 protoplast Anatomy 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000010188 recombinant method Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 210000004994 reproductive system Anatomy 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000001177 retroviral effect Effects 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 238000003345 scintillation counting Methods 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 125000003607 serino group Chemical group [H]N([H])[C@]([H])(C(=O)[*])C(O[H])([H])[H] 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 208000018316 severe headache Diseases 0.000 description 1
- HBMJWWWQQXIZIP-UHFFFAOYSA-N silicon carbide Chemical compound [Si+]#[C-] HBMJWWWQQXIZIP-UHFFFAOYSA-N 0.000 description 1
- 229910010271 silicon carbide Inorganic materials 0.000 description 1
- 238000001542 size-exclusion chromatography Methods 0.000 description 1
- 210000003491 skin Anatomy 0.000 description 1
- 231100000046 skin rash Toxicity 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 210000000278 spinal cord Anatomy 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000000829 suppository Substances 0.000 description 1
- 239000002511 suppository base Substances 0.000 description 1
- 239000000375 suspending agent Substances 0.000 description 1
- 230000035900 sweating Effects 0.000 description 1
- 239000003765 sweetening agent Substances 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 239000006188 syrup Substances 0.000 description 1
- 235000020357 syrup Nutrition 0.000 description 1
- 239000000454 talc Substances 0.000 description 1
- 229910052623 talc Inorganic materials 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- VUYXVWGKCKTUMF-UHFFFAOYSA-N tetratriacontaethylene glycol monomethyl ether Chemical compound COCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCO VUYXVWGKCKTUMF-UHFFFAOYSA-N 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 229940104230 thymidine Drugs 0.000 description 1
- 238000011426 transformation method Methods 0.000 description 1
- 230000010474 transient expression Effects 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 108010080629 tryptophan-leucine Proteins 0.000 description 1
- 108010038745 tryptophylglycine Proteins 0.000 description 1
- 108010044292 tryptophyltyrosine Proteins 0.000 description 1
- 241000701447 unidentified baculovirus Species 0.000 description 1
- 108010073969 valyllysine Proteins 0.000 description 1
- 235000015112 vegetable and seed oil Nutrition 0.000 description 1
- 239000008158 vegetable oil Substances 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
- 238000012800 visualization Methods 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—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
-
- 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
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
- A61K39/42—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum viral
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
-
- 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
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/567—Framework region [FR]
-
- 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
Abstract
본 발명은 뎅기 바이러스 4가지 혈청형의 외막 도메인 Ⅲ에 대한 단일클론 항체 및 이의 용도에 관한 것으로, 본 발명의 단일클론 항체는 뎅기 바이러스 4가지 혈청형 모두를 인식할 수 있기 때문에, 뎅기 바이러스의 감염 진단용 키트에 적용할 수 있으며, 서열분석을 통해 확보된 CDR 서열을 추후 항체 공학 기술을 이용하여 인간화 항체 또는 인간항체로 개발하여 뎅기 바이러스 감염 질환의 치료용 항체로 사용할 수 있을 것이다.The present invention relates to a monoclonal antibody against four types of dengue virus serotypes of the outer membrane domain III and uses thereof. Since the monoclonal antibody of the present invention can recognize all four serotypes of dengue virus, The CDR sequence obtained through sequence analysis can be developed as a humanized antibody or a human antibody by using antibody engineering technology and used as an antibody for treatment of Dengue virus infection disease.
Description
본 발명은 뎅기 바이러스 4가지 혈청형의 외막 도메인 Ⅲ에 대한 단일클론 항체 및 이의 용도에 관한 것이다.The present invention relates to monoclonal antibodies against the outer membrane domain III of four serogroups of dengue virus and their uses.
뎅기열 바이러스는 년간 5천만명에서 1억명이 고통을 받고 있는 세계적인 주요 질환으로서, 년간 25,000명이 사망한다고 알려져 있다. 뎅기(열)은 모기 매개성 질환이며, 감기증상을 보이다가 뎅기출혈열(DHF) 또는 뎅기쇼크증후군(DSS)으로 진행된다. 따라서, 뎅기열 바이러스 감염을 차단하기 위한 치료제의 개발이 무척 중요하나 이 질환의 경우 중복감염(다른 타입의 뎅기 바이러스에 의한 두 번째 감염)이 되면 훨씬 심각한 DHF/DSS 증세를 보이기 때문에, 아직까지는 빠르고 정확한 진단을 통한 환자 증세 완화가 최선의 치료법으로 알려져 있다.Dengue virus is the world's leading disease that affects 50 million to 100 million people annually, and it is known that 25,000 people die each year. Dengue (fever) is a mosquito-borne disease that progresses to dengue haemorrhagic fever (DHF) or dengue shock syndrome (DSS) with cold symptoms. Therefore, the development of therapeutic agents to block dengue virus infections is very important, but in the case of this disease, a double infection (a second infection by another type of Dengue virus) results in a much more severe DHF / DSS condition, Patient symptom relief through diagnosis is known as the best treatment.
뎅기열 바이러스는 4가지 혈청형(serotype)이 있으며, 이 중에서 사람의 항체 형성에 주로 관여하는 외막 단백질(envelope)은 약 70%의 상동성이 있다고 알려져 있다. 이 외막 단백질은 도메인 1(domain 1), 도메인 2(domain 2) 및 도메인 3(domain 3)으로 구별되고, 이 중에서 도메인 3은 숙주세포의 고황화 헤파린(highly sulfated heparan sulfate; HSHS)에 결합하여 바이러스의 세포내 침투를 담당한다고 알려져 있다. 그리고, 외막 단백질의 도메인 3과 도메인 2는 당화(glycosylation)되어 있으며, 중화 에피토프(neutrializing epitope)를 가지고 있어서, 뎅기 바이러스 감염 시, 항체 형성에 주요하게 작용하는 것으로 보고되었다. 하지만, 도메인 1은 주로 외막 단백질의 구조적 역할을 담당하며 dimer 형성에 관여하고, 혈액내에서 낮은 수준의 항체를 형성시킨다고 알려져 있다.Dengue viruses have four serotypes, of which about 70% of the envelope proteins, which are primarily involved in human antibody formation, are known to be homologous. This outer membrane protein is divided into
한편, 한국공개특허 제2014-0019777호에는 '뎅기 바이러스 세로타입 1 E 프로틴에 특이적인 인간 단일클론 항체 및 그들의 용도'가 개시되어 있고, 한국등록특허 제1520084호에는 '뎅기열 바이러스의 표피 단백질 도메인 1에 특이적인 단클론 항체를 포함하는 뎅기열 바이러스 항체의 신속진단키트 및 그 제조 방법'이 개시되어 있으나, 본 발명의 뎅기 바이러스 4가지 혈청형의 외막 도메인 Ⅲ에 대한 단일클론 항체 및 이의 용도에 대해서는 기재된 바가 없다.Korean Patent Laid-Open Publication No. 2014-0019777 discloses' human monoclonal antibodies specific to Dengue virus longitudinal type 1 E protein and their use ', Korean Patent No. 1520084 discloses'
본 발명은 상기와 같은 요구에 의해 도출된 것으로서, 본 발명자들은 뎅기 바이러스 2 혈청형의 외막 도메인 Ⅲ(envelope domain Ⅲ, EDⅢ)을 항원으로 하여, EDⅢ에 특이적인 항체를 생산하는 하이브리도마 클론을 개발하고, 상기 하이브리도마 세포로부터 생산되는 단일클론 항체가, EDⅢ를 특이적으로 검출할 뿐만 아니라, 4가지 혈청형의 뎅기 바이러스 모두를 인식하는 것을 확인함으로써, 본 발명을 완성하였다.Disclosure of the Invention The present invention has been made in view of the above-mentioned needs, and it is an object of the present invention to provide a hybridoma clone producing an antibody specific for EDIII using the envelope domain III (EDIII) of
상기 과제를 해결하기 위해, 본 발명은 뎅기 바이러스 4가지 혈청형의 외막 도메인 Ⅲ(envelope domain Ⅲ, ED Ⅲ)에 결합하는, 마우스로부터 유래된 상보성 결정영역(CDR) 및 프레임 워크 영역(FR)을 포함하는 항체로서, 서열번호 1로 기재된 중쇄 CDR1; 서열번호 2로 기재된 중쇄 CDR2; 및 서열번호 3으로 기재된 중쇄 CDR3을 포함하는 중쇄가변영역과, 서열번호 4로 기재된 경쇄 CDR1; 서열번호 5로 기재된 경쇄 CDR2; 및 서열번호 6으로 기재된 경쇄 CDR3을 포함하는 경쇄가변영역을 포함하는 단일클론 항체 또는 이의 절편을 제공한다.In order to solve the above-mentioned problems, the present invention relates to a method for detecting a complementarity determining region (CDR) derived from a mouse and a framework region (FR) binding to an envelope domain III (ED III) of four serogroups of
또한, 본 발명은 상기 단일클론 항체 또는 이의 절편의 중쇄 가변영역 및 경쇄 가변영역을 코딩하는 폴리뉴클레오티드를 제공한다.The present invention also provides a polynucleotide encoding a heavy chain variable region and a light chain variable region of said monoclonal antibody or fragment thereof.
또한, 본 발명은 상기 폴리뉴클레오티드를 포함하는 발현 벡터를 제공한다.The present invention also provides an expression vector comprising the polynucleotide.
또한, 본 발명은 상기 발현 벡터로 형질전환된 형질전환체를 제공한다.The present invention also provides a transformant transformed with the expression vector.
또한, 본 발명은 상기 형질전환체를 배양하여, 뎅기 바이러스 4가지 혈청형의 외막 도메인 Ⅲ에 결합하는 단일클론 항체의 제조 방법을 제공한다.In addition, the present invention provides a method for producing a monoclonal antibody that binds to the outer membrane domain III of four serogroups of Dengue virus by culturing the transformant.
또한, 본 발명은 상기 단일클론 항체 또는 이의 절편을 유효성분으로 하고, 약학적으로 허용가능한 담체를 포함하는 뎅기 바이러스 감염 질환 예방 또는 치료용 약학 조성물을 제공한다.The present invention also provides a pharmaceutical composition for preventing or treating a dengue virus infection disease, comprising the monoclonal antibody or a fragment thereof as an active ingredient and a pharmaceutically acceptable carrier.
또한, 본 발명은 상기 단일클론 항체 또는 이의 절편을 의심되는 개체의 분리된 생물학적 시료에 처리하여 항원-항체 반응을 통하여 뎅기 바이러스 외막 도메인 Ⅲ 단백질을 검출하는 단계를 포함하는, 뎅기 바이러스 감염을 진단하는 방법을 제공한다.The present invention also relates to a method for diagnosing dengue virus infection comprising the step of treating said monoclonal antibody or fragment thereof with an isolated biological sample of a suspected individual to detect Dengue virus outer membrane domain III protein through an antigen- ≪ / RTI >
또한, 본 발명은 상기 단일클론 항체 또는 이의 절편을 포함하는, 뎅기 바이러스 감염 진단용 조성물을 제공한다.In addition, the present invention provides a composition for diagnosing dengue virus infection comprising the monoclonal antibody or a fragment thereof.
또한, 본 발명은 상기 조성물을 포함하는, 뎅기 바이러스 감염 진단용 키트를 제공한다.The present invention also provides a kit for diagnosing dengue virus infection comprising the composition.
본 발명의 단일클론 항체는 뎅기 바이러스 4가지 혈청형 모두를 인식할 수 있기 때문에, 뎅기 바이러스의 감염 진단용 키트에 적용할 수 있으며, 서열분석을 통해 확보된 CDR 서열을 추후 항체 공학 기술을 이용하여 인간화 항체 또는 인간항체로 개발하여 뎅기 바이러스 감염 질환의 치료용 항체로 사용할 수 있을 것이다.Since the monoclonal antibody of the present invention can recognize all four types of Dengue virus sera, it can be applied to a kit for the diagnosis of dengue virus infection. The CDR sequence obtained through sequence analysis can be humanized Antibodies or human antibodies, and may be used as antibodies for the treatment of Dengue virus infection diseases.
도 1의 (A)는 뎅기 바이러스 2 혈청형의 E 단백질 구조 및 항원으로 사용된 EDⅢ를 나타낸 모식도이며, (B)는 EDⅢmAb-61 단일클론 항체의 반응성을 살펴본 결과이며, (C)는 EDⅢmAb-61 단클론 항체의 아형을 ELISA를 통해 확인한 결과이고, (D)는 4가지 혈청형의 뎅기 바이러스에 대한 EDⅢmAb-61 단일클론 항체의 반응성을 면역 형광 염색을 통해 살펴본 결과이다. rEDⅢ, 재조합 EDⅢ 단백질; cell lysate, DENV-2를 포함하고 있는 세포 용해물; isotype, 일반 생쥐 IgG1을 1차 항체로 사용한 대조군; bar=50㎛.
도 2는 EDⅢmAb-61 단일클론 항체의 중쇄(A) 및 경쇄(B) 아미노산 및 염기서열 정보를 나타낸 것이다.
도 3은 EDⅢmAb-61 단일클론 항체를 포함하는 배양 배지의 rEDⅢ에 대한 반응성을 웨스턴 블랏(A) 및 ELISA(B)를 통해 확인한 결과와, 배양 배지에서 EDⅢmAb-61 단일클론 항체만을 분리 정제하여 뎅기 바이러스 4가지 각 혈청형으로 감염된 Vero 세포에 처리하여 항체 반응성을 면역 형광 염색을 통해 확인한 결과(C)이다.FIG. 1 (A) is a schematic diagram showing the ED protein used as an antigen and the E protein structure of
2 shows the amino acid sequence and base sequence information of the heavy chain (A) and the light chain (B) of the EDII mAb-61 monoclonal antibody.
FIG. 3 shows the results of Western blotting (A) and ELISA (B) showing the reactivity of the EDMI mAb-61 monoclonal antibody-containing culture medium to rEDIII and the EDMI mAb-61 monoclonal antibody alone in the culture medium, (C) results of immunocytochemical staining of antibody reactivity to Vero cells infected with each of four serotypes.
본 발명의 목적을 달성하기 위하여, 본 발명은 뎅기 바이러스 4가지 혈청형의 외막 도메인 Ⅲ에 결합하는 단일클론 항체 또는 이의 절편을 제공한다.In order to accomplish the object of the present invention, the present invention provides a monoclonal antibody or a fragment thereof binding to the outer membrane domain III of four serogroups of dengue virus.
본 발명의 단일클론 항체 또는 이의 절편은 뎅기 바이러스의 외막 도메인 Ⅲ(envelope domain Ⅲ, EDⅢ)에 특이적으로 결합하는 마우스로부터 유래된 상보성 결정영역(complementarity determining region, CDR)과 프레임 워크 영역(framework region, FR)으로 구성되어 있다.The monoclonal antibody or a fragment thereof of the present invention may be used as a complementarity determining region (CDR) and a framework region (CDR) derived from a mouse specifically binding to the envelope domain III (EDIII) of Dengue virus. , FR).
본 발명의 단일클론 항체 또는 이의 절편은, 바람직하게는 서열번호 1로 기재된 중쇄 CDR1; 서열번호 2로 기재된 중쇄 CDR2; 및 서열번호 3으로 기재된 중쇄 CDR3을 포함하는 중쇄가변영역과, 서열번호 4로 기재된 경쇄 CDR1; 서열번호 5로 기재된 경쇄 CDR2; 및 서열번호 6으로 기재된 경쇄 CDR3을 포함하는 경쇄가변영역을 포함하는 것일 수 있으며, 더욱 바람직하게는 서열번호 7의 아미노산 서열로 이루어진 중쇄가변영역 아미노산 서열 및 서열번호 8의 아미노산 서열로 이루어진 경쇄가변영역 아미노산 서열을 포함하는 것일 수 있다.The monoclonal antibody or fragment thereof of the present invention preferably comprises the heavy chain CDR1 as set forth in SEQ ID NO: 1; A heavy chain CDR2 as set forth in SEQ ID NO: 2; And a heavy chain variable region comprising the heavy chain CDR3 as set forth in SEQ ID NO: 3, and a light chain CDR1 as set forth in SEQ ID NO: 4; Light chain CDR2 as set forth in SEQ ID NO: 5; And a light chain variable region comprising the light chain CDR3 of SEQ ID NO: 6, more preferably a heavy chain variable region amino acid sequence consisting of the amino acid sequence of SEQ ID NO: 7 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: Or may comprise an amino acid sequence.
용어 "혈청형(serotype)"은 일반적으로, 뎅기 바이러스 내에 상이한 변이를 지칭한다. 뎅기 바이러스 유전자 군을 포함하는 4가지 상이한 뎅기 바이러스 혈청형(DV1-4)은 아미노산 수준에서 서로 대략 25% 내지 40% 상이하다. 뎅기 바이러스의 4가지 혈청형은 병원성에서 변하지만, 이들 모두 아시아, 아프리카, 중앙아메리카와 남아메리카 지역에 만연하다.The term "serotype" generally refers to different mutations in Dengue virus. Four different dengue virus serotypes (DV1-4), including the dengue virus gene family, are approximately 25% to 40% different from each other at the amino acid level. Four serotypes of Dengue virus vary in pathogenicity, all of which are prevalent in Asia, Africa, Central America, and South America.
본 발명에서 용어 "상보성 결정영역(CDR)"은 항원의 인식에 관여하는 고리모양의 부위로서 이 부위의 서열이 변함에 따라 항체의 항원에 대한 특이성이 결정된다.The term "complementarity determining region (CDR)" in the present invention is an annular region involved in recognition of an antigen, and the sequence of the region is changed, whereby the specificity of the antibody against the antigen is determined.
본 발명에서 용어 "가변영역(variable region)"이란 항원과 특이적으로 결합하는 기능을 수행하면서 서열상의 많은 변이를 보이는 부위를 의미하고, 가변영역에는 상보성 결정영역인 CDR1, CDR2 및 CDR3가 존재한다. 상기 상보성 결정영역 사이에는 프레임 워크 영역(FR) 부분이 존재하여 상보성 결정영역 고리를 지지해주는 역할을 한다.The term "variable region" in the present invention refers to a region showing a large number of mutations in the sequence while specifically binding to an antigen, and complementary determining regions CDR1, CDR2 and CDR3 exist in the variable region . A framework region (FR) portion exists between the complementary crystal regions to support complementary crystal region rings.
본 발명에서 용어 "단일클론 항체"란 단일한 항원성 부위(단일 에피토프)에 대해서 지시되어 이와 특이적인 결합을 하는 단백질 분자를 의미한다. 상기 단일클론항체는 당해 기술 분야에서 잘 알려져 있는 융합 방법(fusion method)에 의해 제조될 수 있다. 일반적으로, 단일클론항체를 분비하는 하이브리도마 세포는 항원 단백질을 주사한 마우스와 같은 면역학적으로 적합한 숙주 동물로부터의 면역 세포와 암 세포주를 융합함으로써 만들어진다. 이런 두 집단의 세포 융합은 폴리에틸렌글리콜과 같이 본 발명이 속하는 기술 분야에 공지되어 있는 방법을 이용하여 융합시키고 항체 생산 세포를 표준적인 배양 방법에 의해 증식시킨다. 한계 희석법(limited dilution)에 의한 서브 클로닝을 실시하여 균일한 세포 집단을 수득하고 난 뒤 항원에 특이적인 항체를 생산할 수 있는 하이브리도마 세포를 시험관 또는 생체 내에서 대량으로 배양한다.As used herein, the term "monoclonal antibody" refers to a protein molecule that is directed against and specifically binds to a single antigenic site (single epitope). The monoclonal antibody may be produced by a fusion method well known in the art. Generally, hybridoma cells that secrete monoclonal antibodies are made by fusing immune cells from cancer cells with immunologically appropriate host animals, such as mice injected with antigen proteins. The cell fusion of these two groups is fused using a method known in the art such as polyethylene glycol, and the antibody producing cells are proliferated by a standard culture method. After subcloning by limited dilution to obtain a uniform cell population, hybridoma cells capable of producing an antigen-specific antibody are cultured in vitro or in vivo.
본 발명에서 항체(antibody)는 전체(whole) 항체 형태를 의미하며, 전체 항체는 2개의 전체 길이의 경쇄 및 2개의 전체 길이의 중쇄를 가지는 구조이며 각각의 경쇄는 중쇄와 디설파이드 결합으로 연결되어 있다.In the present invention, an antibody refers to a whole antibody form, and the whole antibody has a structure having two full-length light chains and two full-length heavy chains, and each light chain is linked to a heavy chain by a disulfide bond .
본 발명의 단일클론 항체는 마우스 항체(mouse antibody), 인간항체(fully human antibody), 인간화 항체(humanized antibody), 키메릭 항체(chimeric antibody) 및 재조합 항체로 이루어진 군으로부터 선택되는 것일 수 있고, 바람직하게는 마우스 항체일 수 있으나, 이에 제한되지 않는다. 본 발명의 마우스 항체는 유전공학적인 기법을 통해 면역화 반응 유발 확률이 적은 인간화 항체 또는 인간항체로 제조하여 치료용 항체로 유용하게 사용될 수 있다.The monoclonal antibody of the present invention may be selected from the group consisting of a mouse antibody, a fully human antibody, a humanized antibody, a chimeric antibody and a recombinant antibody, May be a mouse antibody, but is not limited thereto. The mouse antibody of the present invention can be used as a therapeutic antibody by preparing a humanized antibody or human antibody having a low probability of inducing an immunization reaction through a genetic engineering technique.
본 발명에서 용어 "항체의 절편"이란, 항원 결합 기능을 보유하고 있는 단편을 뜻하며 Fab, F(ab'), F(ab')2 및 Fv 등을 포함한다. 항체 단편 중 Fab는 경쇄 및 중쇄의 가변영역과 경쇄의 불변영역 및 중쇄의 첫 번째 불변영역(CH1)을 가지는 구조로 1개의 항원 결합 부위를 가진다. Fab'는 중쇄 CH1 도메인의 카복시 말단에 하나 이상의 시스테인 잔기를 포함하는 힌지 영역(hinge region)을 가진다는 점에서 Fab와 차이가 있다. F(ab')2 항체는 Fab'의 힌지 영역의 시스테인 잔기가 디설파이드 결합을 이루면서 생성된다. Fv는 중쇄 가변부위 및 경쇄 가변부위만을 가지고 있는 최소의 항체조각으로 Fv 단편을 생성하는 재조합 기술은 국제공개 특허 WO 88/10649 등에 개시되어 있다. 이중쇄 Fv(dsFv)는 디설파이드 결합으로 중쇄 가변부위와 경쇄 가변부위가 연결되어 있고 단쇄 Fv(scFv)는 일반적으로 펩타이드 링커를 통하여 중쇄의 가변영역과 경쇄의 가변영역이 공유결합으로 연결되어 있다. 이러한 항체 단편은 단백질 가수분해 효소를 이용해서 얻을 수 있고(예를 들어, 전체 항체를 파파인으로 제한 절단하면 Fab를 얻을 수 있고, 펩신으로 절단하면 F(ab')2 단편을 얻을 수 있다), 바람직하게는 유전자 재조합 기술을 통하여 제작할 수 있다.The term "antibody fragment" in the present invention means a fragment having an antigen-binding function and includes Fab, F (ab ') 2, F (ab') 2 and Fv. Fabs in the antibody fragment have one antigen-binding site in a structure having a variable region of a light chain and a heavy chain, a constant region of a light chain, and a first constant region (CH1) of a heavy chain. Fab 'differs from Fab in that it has a hinge region containing at least one cysteine residue at the carboxy terminus of the heavy chain CH1 domain. The F (ab ') 2 antibody is produced when the cysteine residue of the hinge region of the Fab' forms a disulfide bond. Recombinant techniques for producing Fv fragments with minimal antibody fragments having only heavy chain variable regions and light chain variable regions are disclosed in WO 88/10649 and others. The double-stranded Fv (dsFv) is linked by a disulfide bond to a light chain variable region and a light chain variable region. A single-chain Fv (scFv) is generally linked to a variable region of a heavy chain and a variable region of a light chain via a peptide linker by a covalent bond. Such an antibody fragment can be obtained using a protein hydrolyzing enzyme (for example, a Fab can be obtained by restriction of the whole antibody to papain, and F (ab ') 2 fragment can be obtained by cleavage with pepsin) Preferably, it can be produced through gene recombination technology.
본 발명의 단일클론 항체 또는 이의 절편은 특별히 이에 제한되지 않으나, 투여된 생체 내에서의 체류시간을 증진시키기 위하여, 당화(glycosylation) 및/또는 페길화(PEGylation)될 수 있다.The monoclonal antibody or a fragment thereof of the present invention is not particularly limited but may be glycosylated and / or PEGylated in order to improve the residence time in a living body.
본 발명의 용어 "당화(glycosylation)"는 글리코실기를 단백질에 전위시키는 가공방법을 의미한다. 상기 당화는 글리코실 전달효소에 의해 글리코실기가 표적 단백질의 세린, 트레오닌, 아스파라긴 또는 히드록실리신 잔기에 결합되어 수행되는데, 상기 당화된 단백질은 생체조직의 구성물질로서 사용될 수 있을 뿐만 아니라, 세포표면에서 세포인식에도 중요한 역할을 수행한다. 따라서, 본 발명에서는 단일클론 항체 또는 이의 절편의 당화 또는 상기 당화의 패턴을 변화시켜서 항체의 효과를 향상시킬 수 있다.The term "glycosylation " of the present invention means a processing method in which a glycosyl group is transferred to a protein. The glycation is carried out by a glycosyltransferase in which a glycosyl group is bonded to a serine, threonine, asparagine or hydrosilicon residue of a target protein. The glycated protein can be used not only as a constituent material of a living tissue, It also plays an important role in cell recognition on the surface. Therefore, in the present invention, the effect of the antibody can be improved by changing the glycosylation or glycosylation pattern of the monoclonal antibody or a fragment thereof.
본 발명의 용어 "페길화(PEGlation)"는 상기 단일클론 항체 또는 이의 절편에 폴리에틸렌글리콜을 도입함으로써, 항체의 혈중 체류시간을 향상시키는 가공방법을 의미한다. 구체적으로, 폴리에틸렌글리콜로 고분자 나노 입자를 페길화하는 것에 의해, 나노 입자의 표면의 친수성이 증가되며 병원균, 노폐물 및 외부 유입 물질을 포식하고 소화시키는 인체 내의 대식세포(macrophage) 등을 포함하는 면역 기능으로부터의 인식을 방지하는 소위 스텔스 효과(stealth effect)를 통한 신체 내에서의 빠른 분해가 방지될 수 있다. 따라서, 상기 페길화에 의하여 항체의 혈중 체류 시간이 향상될 수 있다. 본 발명에서 사용되는 페길화는 히알루론산의 카르복실 그룹과 폴리에틸렌글리콜의 아민 그룹의 결합에 의해 아미드 그룹을 형성하는 방법으로 형성될 수 있으나, 이에 제한되지 않으며 다양한 방법으로 페길화를 수행할 수 있다. 이때, 사용되는 폴리에틸렌글리콜은 특별히 이에 제한되지 않으나, 바람직하게는 100 내지 1,000 사이의 분자량을 갖고, 선형 또는 가지형의 구조를 가지는 것을 사용함이 바람직하다.The term "pegylation " of the present invention means a processing method for improving the retention time of the antibody in blood by introducing polyethylene glycol into the monoclonal antibody or a fragment thereof. Specifically, by pegylating the polymer nanoparticles with polyethylene glycol, the hydrophilicity of the surface of the nanoparticles is increased, and the immune function including macrophages in the human body, which digests and extinguishes pathogens, waste materials, Rapid dissolution in the body through a so-called stealth effect that prevents recognition from the body can be prevented. Therefore, the retention time of the antibody in the blood can be improved by the pegylation. The pegylation used in the present invention may be formed by a method of forming an amide group by bonding a carboxyl group of hyaluronic acid with an amine group of polyethylene glycol, but the present invention is not limited thereto and pegylation can be carried out by various methods . The polyethylene glycol to be used is not particularly limited, but preferably has a molecular weight of 100 to 1,000 and has a linear or branched structure.
상기 당화 및/또는 페길화는 본 발명의 항체의 기능을 유지하는 한 당업계의 공지된 방법에 의해 다양한 당화 및/또는 페길화 패턴이 변형될 수 있고, 본 발명의 항체는 다양한 당화 및/또는 페길화 패턴이 변형된 변이 단일클론 항체 또는 이의 절편을 모두 포함한다.The saccharification and / or pegylation may be modified by a method known in the art so long as the function of the antibody of the present invention is maintained, and the antibody of the present invention may be modified by various saccharification and / The pegylation pattern includes both modified mutated monoclonal antibodies or fragments thereof.
본 발명은 또한, 상기 단일클론 항체 또는 이의 절편의 중쇄 가변영역 및 경쇄 가변영역을 코딩하는 폴리뉴클레오티드를 제공한다. 바람직하게는, 상기 중쇄 가변영역을 코딩하는 폴리뉴클레오티드는 서열번호 9로 기재된 염기서열이고, 상기 경쇄 가변영역을 코딩하는 폴리뉴클레오티드는 서열번호 10의 염기서열이다.The present invention also provides a polynucleotide encoding a heavy chain variable region and a light chain variable region of said monoclonal antibody or fragment thereof. Preferably, the polynucleotide encoding the heavy chain variable region is the nucleotide sequence shown in SEQ ID NO: 9, and the polynucleotide encoding the light chain variable region is the nucleotide sequence shown in SEQ ID NO: 10.
본 발명의 단일클론 항체 또는 이의 절편의 경쇄 및 중쇄를 암호화하는 폴리뉴클레오티드는 코돈의 축퇴성(degeneracy)으로 인하여 또는 상기 인간항체의 경쇄 및 중쇄 또는 그의 단편을 발현시키고자 하는 생물에서 선호되는 코돈을 고려하여, 코딩영역으로부터 발현되는 항체의 경쇄 및 중쇄의 아미노산 서열을 변화시키지 않는 범위 내에서 코딩영역에 다양한 변형이 이루어질 수 있고, 코딩영역을 제외한 부분에서도 유전자의 발현에 영향을 미치지 않는 범위 내에서 다양한 변형 또는 수식이 이루어질 수 있으며, 그러한 변형 유전자 역시 본 발명의 범위에 포함됨을 당업자는 잘 이해할 수 있을 것이다. 즉, 본 발명의 폴리뉴클레오티드는 이와 동등한 활성을 갖는 단백질을 코딩하는 한, 하나 이상의 핵산 염기가 치환, 결실, 삽입 또는 이들의 조합에 의해 변이될 수 있으며, 이들 또한 본 발명의 범위에 포함된다. 이러한 폴리뉴클레오티드의 서열은 단쇄 또는 이중쇄일 수 있으며, DNA 분자 또는 RNA(mRNA) 분자일 수 있다.Polynucleotides encoding the light and heavy chains of the monoclonal antibodies or fragments thereof of the present invention are useful for the detection of the desired codon in the organism to which the light chain and heavy chain of the human antibody or fragments thereof are to be expressed due to degeneracy of the codon Various modifications can be made to the coding region within a range that does not change the amino acid sequence of the light chain and the heavy chain of the antibody expressed from the coding region, and within a range that does not affect the expression of the gene in the portion other than the coding region It will be appreciated by those skilled in the art that various modifications and variations can be made and that such modified genes are also included in the scope of the present invention. That is, as long as the polynucleotide of the present invention encodes a protein having equivalent activity, one or more nucleotide bases may be mutated by substitution, deletion, insertion, or a combination thereof, and these are also included in the scope of the present invention. The sequence of such polynucleotides may be short or double-stranded, and may be a DNA molecule or RNA (mRNA) molecule.
본 발명은 또한, 상기 단일클론 항체 또는 이의 절편의 중쇄 및 경쇄 가변영역을 코딩하는 폴리뉴클레오티드를 포함하는 발현 벡터를 제공한다.The present invention also provides an expression vector comprising a polynucleotide encoding the heavy chain and light chain variable regions of said monoclonal antibody or fragment thereof.
본 발명에서 용어 "발현 벡터"란 적당한 숙주세포에서 목적 단백질을 발현할 수 있는 발현 벡터로서, 유전자 삽입물이 발현되도록 작동가능하게 연결된 필수적인 조절 요소를 포함하는 유전자 작제물을 말한다. 본 발명에서 "작동가능하게 연결된(operably linked)"이란 일반적 기능을 수행하도록 핵산 발현조절 서열과 목적하는 단백질을 코딩하는 폴리뉴클레오티드가 기능적으로 연결되어 있는 것을 말한다. 발현 벡터와의 작동적 연결은 당해 기술분야에서 잘 알려진 유전자 재조합 기술을 이용하여 제조할 수 있으며, 부위-특이적 DNA 절단 및 연결은 당해 기술 분야에서 일반적으로 알려진 효소 등을 사용하여 용이하게 할 수 있다.The term "expression vector" in the present invention refers to an expression vector capable of expressing a desired protein in a suitable host cell, including a necessary regulatory element operably linked to the expression of the gene insert. In the present invention, "operably linked" refers to a functionally linked polynucleotide encoding a desired protein with a nucleic acid expression control sequence to perform a general function. The operative linkage with an expression vector can be made using genetic recombination techniques well known in the art, and site-specific DNA cleavage and linkage can be facilitated using enzymes generally known in the art have.
본 발명의 적합한 발현 벡터는 프로모터, 개시코돈, 종결코돈, 폴리아데닐화 시그널 및 인핸서 같은 발현 조절 엘리먼트 외에도 막 표적화 또는 분비를 위한 시그널 서열을 포함할 수 있다. 개시 코돈 및 종결 코돈은 일반적으로 면역원성 표적 단백질을 코딩하는 뉴클레오타이드 서열의 일부로 간주되며, 유전자 작제물이 투여되었을 때 개체에서 반드시 작용을 나타내야 하며 코딩 서열과 인프레임(in frame)에 있어야 한다. 일반 프로모터는 구성적 또는 유도성일 수 있다. 원핵 세포에는 lac, tac, T3 및 T7 프로모터가 있으나 이에 제한되지는 않는다. 진핵세포에는 원숭이 바이러스 40(SV40) 프로모터, 마우스 유방종양 바이러스(MMTV) 프로모터, 사람 면역 결핍 바이러스(HIV) 프로모터, HIV의 긴 말단 반복부(LTR) 프로모터, 몰로니 바이러스 프로모터, 시토메갈로바이러스(CMV) 프로모터, 엡스타인바 바이러스(EBV) 프로모터, 로우스 사코마 바이러스(RSV) 프로모터 뿐만 아니라, β-액틴 프로모터, 사람 헤모글로빈, 사람 근육 크레아틴, 사람 메탈로티오네인 유래의 프로모터가 있으나 이에 제한되지는 않는다.Suitable expression vectors of the present invention may include signal sequence for membrane targeting or secretion in addition to expression control elements such as promoter, initiation codon, termination codon, polyadenylation signal and enhancer. The initiation codon and termination codon are generally considered to be part of the nucleotide sequence encoding the immunogenic target protein and must be operative in the subject when the gene construct is administered and in the coding sequence and in frame. Generic promoters may be constitutive or inducible. Prokaryotic cells include, but are not limited to, the lac, tac, T3, and T7 promoters. Eukaryotic cells include the monkey virus 40 (SV40) promoter, the mouse breast tumor virus (MMTV) promoter, the human immunodeficiency virus (HIV) promoter, HIV long terminal repeat (LTR) promoter, Moloney virus promoter, But are not limited to, a promoter derived from the human β-actin promoter, the Epstein Barr virus (EBV) promoter, the Roussacomavirus (RSV) promoter as well as the β-actin promoter, human hemoglobin, human muscle creatine and human metallothionein .
발현 벡터는 벡터를 함유하는 숙주 세포를 선택하기 위한 선택성 마커를 포함할 수 있다. 선택마커는 벡터로 형질전환된 세포를 선별하기 위한 것으로, 약물 내성, 영양 요구성, 세포 독성제에 대한 내성 또는 표면 단백질의 발현과 같은 선택가능 표현형을 부여하는 마커들이 사용될 수 있다. 선택제(selective agent)가 처리된 환경에서 선별 마커를 발현하는 세포만 생존하므로 형질전환된 세포가 선별 가능하다.The expression vector may comprise a selectable marker for selecting a host cell containing the vector. Selection markers are for selecting cells transfected with a vector, and markers conferring selectable phenotypes such as drug resistance, nutritional requirement, resistance to cytotoxic agents or expression of surface proteins may be used. In the environment treated with the selective agent, only the cells expressing the selection marker survive, so that the transformed cells can be selected.
또한, 벡터는 복제가능한 발현 벡터인 경우, 복제가 개시되는 특정 폴리뉴클레오티드인 복제원점(replication origin)을 포함할 수 있다. 바이러스 (예를 들어, 바쿨로바이러스) 또는 파지 벡터, 및 레트로바이러스 벡터와 같은 숙주 세포의 게놈내로 삽입될 수 있는 벡터도 사용 가능하다. 전체 항체 또는 항체 단편을 발현하는 벡터는, 경쇄와 중쇄가 하나의 벡터에서 동시에 발현되는 벡터 시스템이거나 또는 경쇄와 중쇄를 각각 별도의 벡터에서 발현시키는 시스템 모두 가능하다. 후자의 경우, 두 벡터는 동시 형질전환(cotransfomation) 및 표적 형질전환(targeted transformation)을 통하여 숙주세포로 도입하고, 경쇄(또는 중쇄)를 함유하는 벡터로 형질전환된 세포를 선별하고 경쇄를 발현하는 선별된 세포를 중쇄(또는 경쇄)를 포함하는 벡터로 다시 형질전환하여 경쇄 및 중쇄 모두를 발현하는 세포를 최종적으로 선별한다. 또한, Fab 형태의 항체를 제작하기 위해서는 인간 경쇄의 가변영역(VL)과 불변영역(CL) 및 인간 중쇄의 가변영역(VH)과 첫 번째 불변 영역 도메인(CH1)의 아미노산을 코딩하는 유전자를 삽입한 벡터를 이용한다.In addition, if the vector is a replicable expression vector, it may contain a replication origin which is a specific polynucleotide at which replication is initiated. Vectors that can be inserted into the genome of a host cell, such as a virus (e. G., Baculovirus) or a phage vector, and a retroviral vector, may also be used. A vector expressing an entire antibody or an antibody fragment can be a vector system in which a light chain and a heavy chain are simultaneously expressed in one vector or a system in which a light chain and a heavy chain are separately expressed in separate vectors. In the latter case, both vectors are introduced into host cells via cotransfomation and targeted transformation, and the cells transformed with the vector containing the light chain (or heavy chain) are screened and the light chain The selected cells are again transformed into a vector containing the heavy chain (or light chain) to finally select cells expressing both light and heavy chains. In order to produce a Fab-type antibody, a gene encoding the amino acid of the variable region (VL) and the constant region (CL) of the human light chain and the variable region (VH) of the human heavy chain and the first constant domain (CH1) One vector is used.
본 발명은 또한, 상기 발현 벡터로 형질전환된 형질전환체를 제공한다.The present invention also provides a transformant transformed with said expression vector.
상기 벡터의 적합한 숙주세포는 에스케리치아 콜라이(Escherichia coli), 바실러스 서브틸리스(Bacillus subtilis), 스트렙토마이세스 속(Streptomyces sp.), 슈도모나스 속(Pseudomonas sp.), 프로테우스 미라빌리스(Proteus mirabilis) 또는 스타필로코쿠스 속(Staphylococcus sp.)과 같은 원핵 세포일 수 있다. 또한, 아스퍼질러스 속(Aspergillus sp.)과 같은 진균, 피치아 파스토리스(Pichia pastoris), 사카로마이세스 세레비지애(Saccharomyces cerevisiae), 쉬조사카로마세스 속(Schizosaccharomyces sp.) 및 뉴로스포라 크라사(Neurospora crassa) 같은 효모, 그 밖의 하등 진핵 세포 및 곤충으로부터의 세포와 같은 고등 진핵생물의 세포와 같은 진핵 세포일 수 있다. 또한 식물, 포유동물로부터 유래할 수 있다. 바람직하게는, 원숭이 신장 세포 7(COS7), NSO 세포, SP2/0, 차이니즈 햄스터 난소 세포(CHO), W138, 어린 햄스터 신장(BHK) 세포, MDCK, 골수종 세포주, HuT 78 세포 및 HEK 293 세포를 포함하나 이에 한정되지 않는다.Suitable host cells of such vectors include, but are not limited to, Escherichia coli , Bacillus subtilis , Streptomyces sp., Pseudomonas sp., Proteus mirabilis ) Or Staphylococcus sp., Or a prokaryotic cell such as Staphylococcus sp. In addition, fungi such as Aspergillus sp., Fungi such as Pichia pastoris), Celebi as Saccharomyces My jiae access (Saccharomyces eukaryotic cells such as cells of higher eukaryotes such as yeasts such as S. cerevisiae , Schizosaccharomyces sp. and Neurospora crassa , and other sub-eukaryotic cells and cells from insects have. It can also be derived from plants and mammals. Preferably, monkey kidney cells 7 (COS7), NSO cells, SP2 / 0, Chinese hamster ovary cells (CHO), W138, young hamster kidney (BHK) cells, MDCK, myeloma cell lines, HuT 78 cells and HEK 293 cells But is not limited thereto.
본 발명에서 "숙주세포로의 형질 전환"은 핵산을 유기체, 세포, 조직 또는 기관에 도입하는 어떤 방법도 포함되며 당 분야에서 공지된 바와 같이 숙주 세포에 따라 적합한 표준 기술을 선택하여 수행할 수 있다. 이런 방법에는 전기충격유전자전달법(electroporation), 원형질 융합, 인산 칼슘(CaPO4) 침전, 염화 칼슘(CaCl2) 침전, 실리콘 카바이드 섬유 이용한 교반, 아그로박테리아 매개된 형질전환, PEG, 덱스트란 설페이트, 리포펙타민(lipofectamine) 및 건조/억제 매개된 형질전환 방법 등이 포함되나, 이에 제한되지 않는다.In the present invention, "transformation into a host cell " includes any method of introducing a nucleic acid into an organism, cell, tissue or organ, and can be carried out by selecting a suitable standard technique depending on the host cell as is known in the art . Such methods include electroporation, protoplast fusion, calcium phosphate (CaPO 4 ) precipitation, calcium chloride (CaCl 2 ) precipitation, agitation with silicon carbide fibers, Agrobacterium mediated transformation, PEG, dextran sulfate, But are not limited to, lipofectamine and dry / inhibition-mediated transformation methods, and the like.
본 발명은 또한, 본 발명의 형질전환체를 배양하여, 뎅기 바이러스 4가지 혈청형의 외막 도메인 Ⅲ에 결합하는 단일클론 항체의 제조 방법을 제공한다. 구체적으로는, (a) 본 발명의 형질전환체를 배양하여 배양액을 제조하는 단계; 및 (b) 상기 (a) 단계의 배양액으로부터 본 발명의 단일클론 항체 또는 이의 절편을 정제하는 단계를 포함하는 것일 수 있다.The present invention also provides a method for producing a monoclonal antibody that binds to the outer membrane domain III of four serogroups of dengue virus by culturing the transformant of the present invention. Specifically, (a) a step of culturing the transformant of the present invention to prepare a culture solution; And (b) purifying the monoclonal antibody or a fragment thereof of the present invention from the culture medium of step (a).
상기 항체 제조에서 형질전환체의 배양은 당업계에 알려진 적당한 배지와 배양조건에 따라 이루어질 수 있다. 이러한 배양과정은 당업자라면 선택되는 균주에 따라 용이하게 조정하여 사용할 수 있다.The culture of the transformant in the production of the antibody may be performed according to a suitable culture medium and culture conditions known in the art. Such a culturing process can be easily adjusted according to the strain selected by those skilled in the art.
형질전환체를 배양하여 수득한 항체는 정제하지 않은 상태로 사용될 수 있으며 추가로 다양한 통상의 정제 방법, 예를 들면 투석, 염 침전, 크로마토그래피 등을 이용할 수 있으며, 이들을 단독 또는 조합하여 이용할 수 있다. 그 중에서 크로마토그래피가 가장 많이 사용되며, 이온교환 크로마토그래피, 크기배제 크로마토그래피, 친화성 크로마토그래피 등이 있다.The antibody obtained by culturing the transformant can be used in an unpurified state. Further, various conventional purification methods, such as dialysis, salt precipitation, chromatography, etc., can be used alone or in combination . Among them, chromatography is most widely used, and there are ion exchange chromatography, size exclusion chromatography, affinity chromatography and the like.
상기 방법으로 제작된 항체는 항원에 대한 친화도(affinity)가 증가된 항체이다. 용어 "친화도"는 항원의 특정부위를 특이적으로 인식하고 결합하는 능력으로, 항체의 항원에 대한 특이성과 함께 고도의 친화도는 면역 반응에서 중요한 요소이다.The antibody produced by the above method is an antibody having an increased affinity to an antigen. The term "affinity" is the ability to specifically recognize and bind to a specific region of an antigen, with high specificity for the antigen, as well as specificity for the antigen, is an important factor in the immune response.
또한, 본 발명은 본 발명의 단일클론 항체 또는 이의 절편을 유효성분으로 하고, 약학적으로 허용가능한 담체를 포함하는 뎅기 바이러스 감염 질환 예방 또는 치료용 약학 조성물을 제공한다.The present invention also provides a pharmaceutical composition for preventing or treating a dengue virus infection disease, comprising a monoclonal antibody or a fragment thereof of the present invention as an active ingredient and a pharmaceutically acceptable carrier.
본 발명의 항체는 뎅기 바이러스의 4가지 혈청형 모두의 외막 도메인 Ⅲ에 결합하므로, 뎅기 바이러스의 침입을 중화시켜 뎅기 바이러스 감염 질환의 치료를 가져올 수 있다. 상기 단일클론 항체 또는 이의 절편에 대해서는 전술한 바와 같다.Since the antibody of the present invention binds to the outer membrane domain III of all four serotypes of Dengue virus, it can neutralize the invasion of Dengue virus and can bring about the treatment of Dengue virus infection disease. The above monoclonal antibody or a fragment thereof is as described above.
본 발명에서 상기 뎅기 바이러스 감염 질환은 뎅기열(dengue fever) 또는 뎅기출혈열(dengue hemorrhagic fever)을 포함할 수 있다. 뎅기열은 뎅기 바이러스가 사람에게 감염되어 생기는 병으로 고열을 동반하는 급성 열성 질환으로, 뎅기 바이러스를 가지고 있는 모기가 사람을 무는 과정에서 전파된다. 뎅기열의 증상은 갑작스럽게 고열이 나서 발열은 3~5일간 계속되고, 심한 두통, 근육통, 관절통, 식욕부진이 생긴다. 초기에 때로 신체 전반에 붉은 반점이 나타난다. 열이 떨어지면서 온 몸에 피부 발진이 1~5일간 계속되는데, 초기에는 얼굴, 목 및 가슴 부위에 좁쌀 모양의 발진이 일시적으로 나타나다가 3~4일째에 가슴과 몸통에서 시작하여 팔다리와 얼굴로 퍼지게 된다. 전신의 림프절이 커지지만 간이나 비장은 촉진되지 않는다. 코피나 잇몸 출혈 등의 경미한 출혈이 질병 경과 중에 나타난다. 성인의 경우 혈변을 보거나 월경과다, 목 부위의 림프절이 붓는 증상이 나타나기도 한다. 뎅기출혈열은 뎅기열의 심한 형태로, 이 경우 환자는 열이 떨어지면서 일시적으로 호전되는 것처럼 보이다가 상태가 급속히 악화되는 양상을 보인다. 매우 심한 쇠약감이나 불안증세가 생기고, 식은땀이 나며, 입 주위가 파랗게 되기도 한다. 가슴의 늑막에 물이 차고, 배에 물이 차는 복수가 생겨서 배가 불러지는 현상이 생길 수도 있다. 뎅기쇼크 증후군이 계속되면 장에서 출혈이 생겨 혈변이 나타난다. 이 경우에는 병의 경과 및 치료 결과가 좋지 않아 사망할 확률이 40~50%에 달하지만, 적극적인 중환자 치료를 받으면 사망률을 낮출 수 있다.In the present invention, the dengue virus infection disease may include dengue fever or dengue hemorrhagic fever. Dengue fever is a disease caused by dengue virus infection in humans. It is an acute febrile disease accompanied by high fever. Symptoms of dengue fever occur suddenly and fever lasts for 3 to 5 days, with severe headaches, muscle aches, arthralgia, and anorexia. Initially, red spots sometimes appear throughout the body. As the fever falls, the skin rash continues for 1 to 5 days throughout the body. Initially, the rash appears on the face, neck and chest area temporarily, then starts on the 3rd and 4th day from the chest and torso, . The lymph nodes of the whole body are enlarged, but the liver and spleen are not promoted. Slight bleeding, such as nosebleeds or bleeding from the gums, occurs during the course of the disease. Adults may have stools, excessive menstrual flow, swelling of the lymph nodes in the neck. Dengue haemorrhagic fever is a severe form of dengue fever in which the patient appears to be temporarily improving as the fever falls, and the condition is rapidly deteriorating. Very severe feelings of anxiety, anxiety, cold sweating, and blue around the mouth. The pleasure of the chest may be filled with water, and the abdomen of the abdomen may develop. Dengue shock syndrome continues, bleeding occurs in the intestines and blood stains appear. In this case, the probability of death is 40 to 50% due to poor outcome and treatment outcome, but if the patient receives active intensive care, the mortality rate can be lowered.
본 발명에서 제공하는 상기 약학 조성물은 약학적으로 허용 가능한 담체를 포함할 수 있다. 본 발명에서 용어, "약학적으로 허용 가능한 담체"란 생물체를 자극하지 않고 투여 화합물의 생물학적 활성 및 특성을 저해하지 않는 담체 또는 희석제를 말한다. 액상 용액으로 제제화되는 조성물에 있어서 약학적으로 허용 가능한 담체로는, 멸균 및 생체에 적합한 것으로서, 식염수, 멸균수, 완충식염수, 알부민 주사용액, 덱스트로즈 용액, 말토덱스트린 용액, 글리세롤 및 이들 성분 중 1 성분 이상을 혼합하여 사용할 수 있으며, 필요에 따라 항산화제, 완충액, 정균제 등 다른 통상의 첨가제를 첨가할 수 있다. 또한 희석제, 분산제, 계면활성제, 결합제 및 윤활제를 부가적으로 첨가하여 수용액, 현탁액, 유탁액 등과 같은 주사용 제형, 환약, 캡슐, 과립 또는 정제로 제제화할 수 있다.The pharmaceutical compositions provided herein may comprise a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not irritate the organism and does not interfere with the biological activity and properties of the administered compound. Examples of the pharmaceutically acceptable carrier for the composition to be formulated into a liquid solution include sterilized and suitable for the living body such as saline, sterilized water, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, One or more components may be mixed and used. If necessary, other conventional additives such as an antioxidant, a buffer, and a bacteriostatic agent may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.
본 발명의 상기 약학 조성물은 경구 또는 비경구의 여러가지 제형일 수 있다. 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다. 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 하나 이상의 화합물에 적어도 하나 이상의 부형제 예를 들면, 전분, 탄산칼슘, 수크로오스(sucrose) 또는 락토오스(lactose), 젤라틴 등을 섞어 조제된다. 또한 단순한 부형제 이외에 스테아린산 마그네슘, 탈크 등과 같은 윤활제들도 사용된다. 경구투여를 위한 액상제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데 흔히 사용되는 단순 희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조제제, 좌제가 포함된다. 비수성용제, 현탁용제로는 프로필렌글리콜(propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테로 등이 사용될 수 있다. 좌제의 기제로는 위텝솔(witepsol), 마크로골, 트윈(tween) 61, 카카오지, 라우린지, 글리세로젤라틴 등이 사용될 수 있다.The pharmaceutical composition of the present invention may be various oral or parenteral formulations. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules, and the like, which may contain one or more excipients such as starch, calcium carbonate, sucrose or lactose lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate, talc, and the like are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used. have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the non-aqueous solvent and the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. Examples of the suppository base include witepsol, macrogol,
본 발명은 또한, 상기 단일클론 항체 또는 이의 절편을 의심되는 개체의 분리된 생물학적 시료에 처리하여 항원-항체 반응을 통하여 뎅기 바이러스 외막 도메인 Ⅲ 단백질을 검출하는 단계를 포함하는, 뎅기 바이러스 감염을 진단하는 방법을 제공한다.The present invention is also directed to a method for diagnosing dengue virus infection comprising the step of treating said monoclonal antibody or fragment thereof with a separate biological sample of a suspected individual to detect Dengue virus outer membrane domain III protein through an antigen- ≪ / RTI >
상기 단일클론 항체 또는 이의 절편에 대해서는 전술한 바와 같다.The above monoclonal antibody or a fragment thereof is as described above.
상기 뎅기 바이러스 감염을 진단하는 방법은 본 발명의 뎅기 바이러스 4가지 혈청형의 외막 도메인 Ⅲ에 결합하는 단일클론 항체를 뎅기 바이러스 감염이 의심되는 개체의 분리된 생물학적 시료와 반응시키고, 항원-항체 복합체 형성을 검출함으로써 확인할 수 있다.The method for diagnosing Dengue virus infection comprises reacting a monoclonal antibody binding to the outer membrane domain III of the four serogroups of the present invention with an isolated biological sample suspected of having dengue virus infection and forming an antigen- As shown in FIG.
본 발명에서 용어 "생물학적 시료"란, 조직, 세포, 전혈, 혈청, 혈장, 조직 부검 시료(뇌, 피부, 림프절, 척수 등), 세포 배양 상등액, 파열된 진핵세포 및 세균 발현계 등을 들 수 있으나, 이에 제한되는 것은 아니다. 이들 생물학적 시료를 조작하거나 조작하지 않은 상태로 본 발명의 항체와 반응시켜 뎅기 바이러스 외막 도메인 Ⅲ의 존재 또는 뎅기 바이러스 감염의 유무를 확인할 수 있다.The term "biological sample" in the present invention includes tissues, cells, whole blood, serum, plasma, tissue autopsy samples (brain, skin, lymph nodes, spinal cord etc.), cell culture supernatant, ruptured eukaryotic cells, But is not limited thereto. These biological samples may be reacted with the antibody of the present invention without manipulating or manipulating them to confirm the presence of Dengue virus outer membrane domain III or the presence of dengue virus infection.
본 발명에서 용어 "항원-항체 복합체"란, 시료 중의 뎅기 바이러스 외막 도메인 Ⅲ 항원과 이를 인지하는 본 발명에 따른 단일클론 항체 또는 이의 절편의 결합물을 의미하며, 이러한 항원-항체 복합체의 형성은 비색법(colormetric method), 전기화학법(electrochemical method), 형광법(fluorimetric method), 발광법(luminometry), 입자계수법(particle counting method), 육안측정법(visual assessment) 및 섬광계수법(scintillation counting method)으로 이루어진 군에서 선택되는 임의의 방법으로 검출할 수 있다. 그러나 반드시 이들로만 제한되지 않고 다양한 응용과 적용이 가능하다.The term "antigen-antibody complex " in the present invention means a conjugate of a Dengue virus outer membrane domain III antigen in the sample and a monoclonal antibody or a fragment thereof recognizing the Dengue virus outer membrane domain III antigen. a colorimetric method, an electrochemical method, a fluorimetric method, a luminometry method, a particle counting method, a visual assessment method, and a scintillation counting method And the like. However, various applications and applications are possible without being limited thereto.
본 발명에서는 항원-항체 복합체를 검출하기 위한 것으로 여러가지 표지체를 사용할 수 있다. 구체적인 예로는 효소, 형광물, 리간드, 발광물, 미소입자, 방사성 동위원소로 이루어진 그룹 중에서 선택될 수 있으며, 반드시 이들로만 한정되는 것은 아니다.In the present invention, various markers can be used for detecting an antigen-antibody complex. Specific examples include, but are not limited to, enzymes, chromophores, ligands, luminescent materials, microparticles, and radioactive isotopes.
검출 표지체로서 사용되는 효소로는 아세틸콜린에스테라제, 알칼라인 포스파타제, β-D-갈락토시다제, 호스래디쉬 퍼옥시다제, β-라타마제 등을 포함하며, 형광물로는 플루오레세인, Eu3+, Eu3+ 킬레이트 또는 크립테이트 등을 포함하며, 리간드로는 바이오틴 유도체 등을 포함하며, 발광물로는 아크리디늄 에스테르, 이소루미놀 유도체 등을 포함하며, 미소입자로는 콜로이드 금, 착색된 라텍스 등을 포함하며, 방사성 동위원소로는 57Co, 3H, 125I, 125I-볼톤(Bonton) 헌터(Hunter) 시약 등을 포함한다.Examples of the enzyme used as the detection label include acetylcholine esterase, alkaline phosphatase,? -D-galactosidase, horseradish peroxidase,? -Lactamase and the like, Phosphorus, Eu 3+ , Eu 3+ chelate or cryptate, etc. Examples of the ligand include biotin derivatives and the like. Emitters include acridinium ester, isoluminol derivatives and the like. Examples of the fine particles include colloid Gold, colored latex and the like, and the radioactive isotopes include 57 Co, 3 H, 125 I, 125 I-Bonton Hunter reagent and the like.
바람직하게는, 항원-항체 복합체를 효소면역흡착법(ELISA)을 이용하여 검출할 수 있다. 효소면역흡착법은 고체 지지체에 부착된 항원을 인지하는 항체를 이용하는 직접적 ELISA, 고체 지지체에 부착된 항원을 인지하는 항체의 복합체에서 포획 항체를 인지하는 표지된 이차 항체를 이용하는 간접적 ELISA, 고체 지지체에 부착된 항체와 항원의 복합체에서 항원을 인지하는 표지된 또 다른 항체를 이용하는 직접적 샌드위치 ELISA, 고체 지지체에 부착된 항체와 항원의 복합체에서 항원을 인지하는 또 다른 항체와 반응시킨 후 이 항체를 인지하는 표지된 이차 항체를 이용하는 간접적 샌드위치 ELISA 등 다양한 ELISA 방법을 포함한다.Preferably, the antigen-antibody complexes can be detected using enzyme immunoassay (ELISA). Enzyme immunosorbent assays include direct ELISA using an antibody recognizing an antigen attached to a solid support, indirect ELISA using a labeled secondary antibody recognizing the capture antibody in a complex of antibodies recognizing an antigen attached to a solid support, attachment to a solid support A direct sandwich ELISA using another labeled antibody that recognizes the antigen in the complex of the antibody and the antigen, a label which recognizes the antibody after reacting with another antibody recognizing the antigen in the complex of the antibody and the antigen attached to the solid support And indirect sandwich ELISA using secondary antibodies.
상기 단일클론 항체 또는 이의 절편은 검출 표지를 가질 수 있으며, 검출 표지를 가지지 않을 경우는 이들 단일클론 항체 또는 이의 절편을 포획할 수 있고, 검출표지를 가지는 또 다른 항체를 처리하여 확인할 수 있다.The monoclonal antibody or fragment thereof may have a detection label, and when it does not have a detection label, it may capture these monoclonal antibodies or fragments thereof and can be identified by treating another antibody having the detection label.
본 발명은 또한, 본 발명의 단일클론 항체 또는 이의 절편을 포함하는, 뎅기 바이러스 감염 진단용 조성물을 제공한다.The present invention also provides a composition for diagnosing dengue virus infection comprising a monoclonal antibody or a fragment thereof of the present invention.
상기 단일클론 항체 또는 이의 절편에 대해서는 전술한 바와 같다. 본 발명의 뎅기 바이러스 외막 도메인 Ⅲ에 결합하는 단일클론 항체 또는 이의 절편을 포함하는 진단용 조성물을 사용하여 뎅기 바이러스 외막 도메인 Ⅲ의 검출 유무와 관련된 뎅기 바이러스 감염을 진단할 수 있다.The above monoclonal antibody or a fragment thereof is as described above. Dengue virus infection associated with the detection of Dengue virus outer membrane domain III can be diagnosed by using a diagnostic composition comprising a monoclonal antibody or a fragment thereof bound to Dengue virus outer membrane domain III of the present invention.
또한, 본 발명은 상기 뎅기 바이러스 감염 진단용 조성물을 포함하는 뎅기 바이러스 감염 진단용 키트를 제공한다.The present invention also provides a diagnostic kit for dengue virus infection comprising the composition for diagnosing dengue virus infection.
상기 조성물에 대해서는 상기에서 설명한 바와 같다. 또한, 뎅기 바이러스 감염 진단용 키트는 분석방법에 적합한 한 종류 또는 그 이상의 다른 구성성분을 가진 조성물, 용액 또는 장치를 더 포함하여 구성될 수 있다.
The above composition is as described above. The kit for diagnosing a dengue virus infection may further comprise a composition, solution or device having one or more other components suitable for the assay method.
이하, 본 발명을 실시예에 의해 상세히 설명한다. 단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 한정되는 것은 아니다.
Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.
재료 및 방법Materials and methods
1. 실험재료1. Experimental material
본 발명에 사용된 모든 시약류들은 시그마 케미컬(미국)로부터 구매하여 사용하였다. BALB/c 생쥐(암컷, 5주령)는 오리엔트 바이오(한국)로부터 구매하였다.
All of the reagents used in the present invention were purchased from Sigma Chemical (USA). BALB / c mice (female, 5 weeks old) were purchased from Orient Bio (Korea).
2. 바이러스 및 세포주2. Viruses and cell lines
환자로부터 분리한 네가지 뎅기 바이러스는 Truong 박사(베트남 국립보건연구원)로부터 제공받았으며, C6/36 세포주는 ATCC(미국)로부터 구매하여 5%(v/v) FBS(소태아혈청, Etobicoke, 캐나다)가 첨가된 최소영양배지를 이용하여 28℃, CO2 조건에서 배양하며 증식시켰다. SP2/0-Ag14, CHO-K1 및 Vero 세포주 역시 ATCC로부터 구매하여 Dulbecco's 최소영양배지와 10% FBS가 첨가된 F-12K 배지를 사용하여 37℃, CO2 조건에서 배양하였다.
The C6 / 36 cell line was purchased from ATCC (USA) and 5% (v / v) FBS (fetal bovine serum, Etobicoke, Canada) was purchased from Dr. Truong (Vietnam National Health Research Institute) The cells were cultured at 28 ℃ and CO 2 with the supplemented minimal nutrient medium. SP2 / 0-Ag14, CHO-K1 and Vero cell lines were also purchased from ATCC and cultured in CO 2 at 37 ° C using Dulbecco's minimal nutrient medium and F-12K medium supplemented with 10% FBS.
3. 재조합 단백질3. Recombinant protein
DENV-2로부터 유래한 재조합 EDⅢ(rEDⅢ) 단백질을 면역원으로 사용하였다. DENV-2의 EDⅢ 유전자는 DENV-2 감염된 C6/36 세포로부터 증폭시키고, pREST 발현 벡터(Novagen, 독일)에 클로닝하였다. rEDⅢ 단백질은 BL21(DE3) E. coli에서 발현시키고, Ni-NTA 친화성 크로마토그래피 시스템(Bio-rad, 미국)을 통해 정제하였다.
Recombinant EDIII (rEDIII) protein derived from DENV-2 was used as an immunogen. The EDIII gene of DENV-2 was amplified from DENV-2 infected C6 / 36 cells and cloned into pREST expression vector (Novagen, Germany). The rEDIII protein was expressed in BL21 (DE3) E. coli and purified through a Ni-NTA affinity chromatography system (Bio-rad, USA).
4. B 세포 하이브리도마 클론의 생성 및 유지4. Production and maintenance of B cell hybridoma clones
rEDⅢ 단백질 100㎍으로 주사 후 한달 후 동량으로 일주일에 한번씩 2주 주사하여 면역화한 생쥐로부터 지라세포를 준비한 후 1㎖의 PEG-1500(Roche Diagnostics GmbH, 독일)를 사용하여 SP2/0-Ag14 골수종 세포주와 융합시켰다. 융합된 세포는 HAT(hypoxanthine-aminopterin-thymidine) 배지에서 선별하였다. EDⅢ 특이적 항체를 분비하는 하이브리도마 세포주는 항원 directed ELISA를 통해 재선별하였으며, 단클론 하이브리도마는 한계 희석법을 통해 획득하였다.
The SP2 / 0-Ag14 myeloma cell line was cultured with 1 ml of PEG-1500 (Roche Diagnostics GmbH, Germany) after preparing the spleen cells from the immunized mice by injecting 100 .mu.g of rEDIII protein one week after the injection, ≪ / RTI > Fused cells were selected on HAT (hypoxanthine-aminopterin-thymidine) medium. Hybridoma cell lines secreting EDII-specific antibodies were re-screened by antigen directed ELISA and monoclonal hybridomas were obtained by limiting dilution.
5. 하이브리도마 클론으로부터 면역글로불린 유전자의 클로닝 및 일과성 발현5. Cloning and transient expression of immunoglobulin genes from hybridomas
하이브리도마 클론 #61의 총 RNA는 RNeasy 미니 키트(Qiagen, 독일)를 사용하여 준비하였고, cDNA 합성은 QuantiTect 역전사 시스템(Qiagen)을 이용하였다. 면역글로불린 경쇄 및 중쇄 유전자는 Ig-Primer 세트(Millipore, 미국)를 사용하여 98℃ 10초, 55℃ 30초, 72℃ 1분의 사이클을 30회 반복하는 조건으로 PCR을 수행하여 증폭하였다. PCR 산물은 pcDNA3.1 포유류 세포 발현 벡터(Invitrogen, 미국)로 서브클로닝하였다. 면역글로불린 유전자를 포함하고 있는 벡터를 lipofectamin 3000(Invitrogen)을 이용하여 CHO-K1 세포에 형질전환하였다. 간단하게, 플라스미드 DNA와 지질(lipofectamin)의 1:1 혼합물을 준비하고, 이를 세포에 처리한 후 1일 후에 배양 배지를 G418 설페이트가 포함된 신선한 배지로 교환하였다. 면역글로불린 유전자를 가지고 있는 CHO-K1을 4일간 배양하고 4℃, 3,000rpm, 10분의 조건으로 원심분리한 상층액을 단백질 G 컬럼(GE Healthcare, 영국)을 사용하여 세포로부터 분비되는 항체들을 분리하였다.
Total RNA of
6. ELISA(Enzyme-linked immunosorbent assay)6. Enzyme-linked immunosorbent assay (ELISA)
rEDⅢ 단백질 또는 항-아형(anti-isotype) 항체로 코팅된 ELISA 플레이트의 각 웰은 3% BSA(소혈청알부민)가 녹아있는 PBS로 블록킹하였다. 그 후 블록킹 용액을 제거하고 웰을 세척한 후, #61 하이브리도마 세포의 배양액을 각 웰에 넣고 반응시킨 후, 붙어있는 항체를 알카라인 포스페이트가 붙어있는 이차 항체와 순차적인 배양을 통해 검출하였다. 최종적으로, p-니트로페닐 포스페이트(p-nitrophenyl phosphate) 용액을 첨가하고, 효소 활성을 ELISA 리더기(Packard Instrument, 미국)로 측정하였다.
Each well of an ELISA plate coated with rEDIII protein or anti-isotype antibody was blocked with PBS in which 3% BSA (bovine serum albumin) was dissolved. Thereafter, the blocking solution was removed and the wells were washed. Then, the culture solution of # 61 hybridoma cells was added to each well and reacted. Then, the attached antibody was detected by sequential culturing with secondary antibody attached with alkaline phosphate. Finally, a solution of p-nitrophenyl phosphate was added and enzyme activity was measured with an ELISA reader (Packard Instrument, USA).
7. 웨스턴 블랏 분석7. Western blot analysis
rEDⅢ 단백질 및 DENV를 포함하고 있는 Vero 세포 용해물을 15% SDS-PAGE 겔에 로딩하여 분리한 후 PVDF 멤브레인에 트랜스퍼하였다. 면역블롯팅(Immunoblotting)은 EDⅢ 특이적 단클론 항체 EDⅢmAb-61, #61 하이브리도마 세포의 배양액, 항-뎅기 바이러스 항체(AbD Serotec, 영국)를 이용하여 수행되었다. 시각화를 위해서, HRP(horseradish perosidase)가 붙어있는 이차항체와 화학발광(chemiluminescence)을 사용하였다.
Vero cell lysate containing rEDIII protein and DENV was loaded on a 15% SDS-PAGE gel, separated and transferred to a PVDF membrane. Immunoblotting was performed using ED III-specific mAb EDII mAb-61, a culture of # 61 hybridoma cells, anti-Dengue virus antibody (AbD Serotec, UK). For visualization, a secondary antibody with HRP (horseradish perosidase) and chemiluminescence were used.
8. 면역 형광 염색(Immunofluorescent staining)8. Immunofluorescent staining.
DENV로 감염된 Vero 세포를 4% 파라포름알데히드로 고정한 후, 3% BSA가 녹아있는 PBS로 블록킹하고, EDⅢmAb-61 단일클론 항체와 반응시킨 후, FITC(fluorescein isothiocyanate)가 결합된 이차 항체(BD Bioscience, 미국)를 반응시켜 세포를 염색하였다. DAPI(Invitrogen)로 핵을 염색한 후 공초점 주사 레이져 현미경(confocal laser scanning microscopy, Carl zeiss, 독일)으로 시료를 분석하였다.
Vero cells infected with DENV were fixed with 4% paraformaldehyde, blocked with PBS containing 3% BSA, reacted with an EDII mAb-61 monoclonal antibody, and then reacted with FITC (fluorescein isothiocyanate) -based secondary antibody (BD Bioscience , USA) were reacted to stain the cells. Nuclei were stained with DAPI (Invitrogen) and analyzed with a confocal laser scanning microscope (Carl Zeiss, Germany).
실시예 1. EDⅢ 특이적 단클론 항체를 생산하는 하이브리도마 클론 #61Example 1. Hybridoma clone producing EDII-specific
뎅기 바이러스의 E 단백질은 세 개의 도메인으로 구성되어 있고, EDⅢ는 297번째 아미노산부터 394번째 아미노산까지이다(도 1A). rEDⅢ 단백질을 준비하기 위해서, EDⅢ 유전자를 pREST 발현 벡터로 클로닝하고, 대장균 시스템에서 발현시킨 후 Ni-NTA 친화성 컬럼으로 분리하였다. rEDⅢ는 폴리히스티딘, T7 유전자 및 Xpress 에피토프를 가진 약 15kDa의 단백질이다. EDⅢ-특이적 단일클론 항체를 생산하기 위해서, rEDⅢ로 면역화한 생쥐로부터 분리한 지라세포를 SP2/0-Ag14 골수종 세포와 융합하여 EDⅢ 특이적 ELISA를 통해 7개의 클론을 선별하였다. 그 중, #61 하이브리도마 클론으로부터 유래한 단일클론 항체가 rEDⅢ 뿐만 아니라, DENV-2를 포함하고 있는 세포 용해물의 E 단백질까지 검출하는 것을 확인하였으며(도 1B), #61 하이브리도마 클론의 항체 아형은 IgG1인 것으로 확인되었다(도 1C). 이에 본 발명에서는 #61 하이브리도마 클론으로부터 유래한 단일클론 항체를 EDⅢmAb-61로 명명하였다. 상기 EDⅢmAb-61 항체의 특징을 분석하기 위해서, 본 발명의 항체가 다른 혈청형의 뎅기 바이러스에 반응하는지 살펴보았다. 면역 형광 염색 결과, EDⅢmAb-61 단일클론 항체가 4가지 혈청형의 뎅기 바이러스 모두를 인식하는 것을 확인하였다(도 1D).The E protein of Dengue virus consists of three domains, EDIII ranging from the 297th amino acid to the 394th amino acid (Figure 1A). To prepare the rEDIII protein, the EDIII gene was cloned into a pREST expression vector, expressed in an E. coli system, and then separated into Ni-NTA affinity columns. rEDIII is a protein of approximately 15 kDa with polyhistidine, T7 gene and Xpress epitope. To produce EDIII-specific monoclonal antibodies, seven clones were selected by EDII-specific ELISA by fusion of SP2 / 0-Ag14 myeloma cells with spleen cells isolated from mice immunized with rEDIII. Among them, it was confirmed that a monoclonal antibody derived from # 61 hybridomal clone detects not only rEDII but also E protein of cell lysate containing DENV-2 (Fig. 1B), and a # 61 hybridoma clone Lt; RTI ID = 0.0 > IgG1 < / RTI > (Figure 1C). In the present invention, the monoclonal antibody derived from the # 61 hybridoma clone was named EDIII mAb-61. In order to analyze the characteristics of the EDII mAb-61 antibody, it was examined whether the antibody of the present invention reacted with other serotype dengue viruses. Immunofluorescent staining confirmed that ED III mAb-61 monoclonal antibody recognizes all four serotypes of dengue virus (Fig. 1D).
상기의 결과들을 통해, 본 발명의 하이브리도마 클론이 생산하는 단일클론 항체가 네 가지 혈청형의 뎅기 바이러스 모두의 EDⅢ에 특이적인 것을 알 수 있었다.
From the above results, it was found that the monoclonal antibody produced by the hybridoma clone of the present invention is specific to EDIII of all four serotype Dengue viruses.
실시예 2. EDⅢmAb-61 단일클론 항체의 서열 분석Example 2. Sequence analysis of EDII mAb-61 monoclonal antibody
EDⅢmAb-61 단일클론 항체의 상보성 결정 영역(complementarity determining region, CDR)을 분석하기 위해서, #61 하이브리도마 클론으로부터 준비된 중쇄 및 경쇄 유전자를 T 벡터로 클로닝하고, 제노텍(한국)에 의뢰하여 서열을 분석하였다. 그 결과, 중쇄 유전자는 19개의 아미노산을 포함하는 신호 펩타이드를 가지고 있음을 알 수 있었고, 가변 영역은 IgBLAST를 통해 분석하였다. 중쇄의 V 유전자는 IGHV1-12*01 생식 계통 유전자(germ line gene)와 매우 유사하였으며, D 유전자는 IGHD2-9*01, IGHD2-2*01 및 IGHD2-7**01 생식 계통 유전자와, 그리고 J 유전자는 IGHJ4*01 생식 계통 유전자와 일치하였다. IMGT/Junction 분석을 통해 예측된 중쇄 CDR3의 아미노산 서열은 CARWLRNYAMDYW이었다. 경쇄 유전자 분석 결과 또한 V 유전자는 IGKVI-110*01 생식 계통 유전자와 J 유전자는 IGKJ2*01 생식 계통 유전자와 일치하였다. 경쇄 CDR3의 아미노산 서열은 CSQSTHDPYTF로 확인되었다(도 2).
To analyze the complementarity determining region (CDR) of the EDII mAb-61 monoclonal antibody, the heavy and light chain genes prepared from the # 61 hybridoma clone were cloned into T vectors and submitted to Genotech (Korea) Respectively. As a result, it was found that the heavy chain gene had a signal peptide containing 19 amino acids, and the variable region was analyzed through IgBLAST. The heavy chain V gene is very similar to the IGHV1-12 * 01 germ line gene, the D gene is associated with the IGHD2-9 * 01, IGHD2-2 * 01 and IGHD2-7 ** 01 reproductive line genes, and The J gene was consistent with the IGHJ4 * 01 reproductive gene. The amino acid sequence of heavy chain CDR3 predicted by IMGT / Junction analysis was CARWLRNYAMDYW. Light chain gene analysis also showed that the V gene was identical to the IGKJ2 * 01 reproductive gene gene in the IGKVI-110 * 01 reproductive system gene and the J gene. The amino acid sequence of light chain CDR3 was identified as CSQSTHDPYTF (Figure 2).
실시예 3. EDⅢmAb-61 단일클론 항체의 뎅기 바이러스 검출 분석Example 3. Detection of dengue virus in EDII mAb-61 monoclonal antibody
EDⅢmAb-61 단일클론 항체의 진정성을 확인하기 위해, 각 중쇄 및 경쇄 유전자를 pcDNA3.1 벡터로 서브클로닝하고 CHO-K1 세포에서 발현시켰다. EDⅢmAb-61 단일클론 항체 유전자를 가지고 있는 CHO 세포의 배양 배지를 이용하여 rEDⅢ 단백질 검출을 웨스턴 블랏(도 3A)과 ELISA(도 3B)로 분석하였다. EDⅢmAb-61 단일클론 항체가 뎅기 바이러스를 검출할 수 있는지 알아보기 위해, 단백질 G 컬럼을 통해 배양 배지로부터 단일클론 항체만 분리하였다. 분리된 EDⅢmAb-61 단일클론 항체를 4가지 혈청형의 뎅기 바이러스 각각에 감염된 Vero 세포에 처리하였을 때, 면역 형광 염색을 통해 EDⅢmAb-61 단일클론 항체가 모든 혈청형의 뎅기 바이러스를 검출하는 것을 확인하였다(도 3C). 종합적으로, 본 발명의 EDⅢmAb-61 단일클론 항체가 EDⅢ에 특이적인 항체임을 알 수 있었다.To confirm the authenticity of the EDII mAb-61 monoclonal antibody, each heavy and light chain gene was subcloned into pcDNA3.1 vector and expressed in CHO-K1 cells. The detection of rEDIII protein was analyzed by Western blotting (FIG. 3A) and ELISA (FIG. 3B) using the culture medium of CHO cells harboring EDII mAb-61 monoclonal antibody gene. To determine if EDII mAb-61 monoclonal antibodies can detect dengue virus, only monoclonal antibodies were isolated from the culture medium via protein G column. When the EDII mAb-61 monoclonal antibody isolated was treated with Vero cells infected with each of the four serotypes of dengue virus, the EDII mAb-61 monoclonal antibody was detected by immunofluorescence staining to detect all serovar dengue viruses (Fig. 3C). Overall, the EDII mAb-61 monoclonal antibody of the present invention was an EDII-specific antibody.
<110> INDUSTRIAL COOPERATION FOUNDATION CHONBUK NATIONAL UNIVERSITY <120> Monoclonal antibody against envelope domain III of four serotype dengue virus and uses thereof <130> PN15453 <160> 10 <170> KopatentIn 2.0 <210> 1 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> VH CDR1 <400> 1 Gly Tyr Thr Phe Thr Ser Tyr Asn 1 5 <210> 2 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> VH CDR2 <400> 2 Ile Tyr Pro Gly Asn Gly Tyr Thr Ser 1 5 <210> 3 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> VH CDR3 <400> 3 Cys Ala Arg Trp Leu Arg Asn Tyr Ala Met Asp Tyr Trp 1 5 10 <210> 4 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> VL CDR1 <400> 4 Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu 1 5 10 <210> 5 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> VL CDR2 <400> 5 Val Ser Asn 1 <210> 6 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> VL CDR3 <400> 6 Cys Ser Gln Ser Thr His Asp Pro Tyr Thr Phe 1 5 10 <210> 7 <211> 140 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 7 Met Gly Trp Ser Tyr Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys 20 25 30 Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Ser Tyr Asn Met His Trp Val Lys Gln Thr Pro Gly Gln Gly Leu 50 55 60 Glu Trp Ile Gly Ala Ile Tyr Pro Gly Asn Gly Tyr Thr Ser Tyr Asn 65 70 75 80 Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Arg Ser Ser Ser 85 90 95 Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val 100 105 110 Tyr Phe Cys Ala Arg Trp Leu Arg Asn Tyr Ala Met Asp Tyr Trp Gly 115 120 125 Gln Gly Thr Ser Val Thr Val Ser Ser Ala Lys Thr 130 135 140 <210> 8 <211> 140 <212> PRT <213> Artificial Sequence <220> <223> VL <400> 8 Met Arg Ala Pro Ala Gln Phe Phe Gly Phe Leu Leu Leu Trp Phe Pro 1 5 10 15 Ala Ser Ser Ser Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro 20 25 30 Val Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser 35 40 45 Leu Val His Ser Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys 50 55 60 Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe 65 70 75 80 Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 85 90 95 Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe 100 105 110 Cys Ser Gln Ser Thr His Asp Pro Tyr Thr Phe Gly Gly Gly Thr Lys 115 120 125 Leu Glu Ile Lys Arg Ala Asp Ala Ala Pro Thr Val 130 135 140 <210> 9 <211> 420 <212> DNA <213> Artificial Sequence <220> <223> VH <400> 9 atgggatgga gctatatcat cctcttcttg gtagcaacag ctacaggtgt ccactcccag 60 gtgcaactgc agcagcctgg ggctgagctg gtgaagcctg gggcctcagt gaagatgtcc 120 tgcaaggctt ctggctacac atttaccagt tacaatatgc actgggtaaa gcagacacct 180 ggacagggcc tggaatggat tggagctatt tatccaggaa atggttatac ttcctacaat 240 cagaagttca aaggcaaggc cacattgact gcagacagat cctccagcac agcctacatg 300 cagctcagca gcctgacatc tgaggactct gcggtctatt tctgtgcaag atggttacga 360 aactatgcta tggactactg gggtcaagga acctcagtca ccgtctcctc agccaaaacg 420 420 <210> 10 <211> 420 <212> DNA <213> Artificial Sequence <220> <223> VL <400> 10 atgagggccc ctgctcagtt ttttgggttc ttgttgctct ggtttccagc ttccagcagt 60 gatgttgtga tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 120 atctcttgca gatctagtca gagccttgta cacagtaatg gaaacaccta tttacattgg 180 tacctgcaga agccaggcca gtctccaaag ctcctgatct acaaagtttc caaccgattt 240 tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc 300 agcagagtgg aggctgagga tctgggagtt tatttctgct ctcaaagtac acatgatccg 360 tacacgttcg gaggggggac caagctggaa ataaaacggg ctgatgctgc accaactgta 420 420 <110> INDUSTRIAL COOPERATION FOUNDATION CHONBUK NATIONAL UNIVERSITY <120> Monoclonal antibody against envelope domain III of four serotype dengue viruses and uses thereof <130> PN15453 <160> 10 <170> Kopatentin 2.0 <210> 1 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> VH CDR1 <400> 1 Gly Tyr Thr Phe Thr Ser Tyr Asn 1 5 <210> 2 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> VH CDR2 <400> 2 Ile Tyr Pro Gly Asn Gly Tyr Thr Ser 1 5 <210> 3 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> VH CDR3 <400> 3 Cys Ala Arg Trp Leu Arg Asn Tyr Ala Met Asp Tyr Trp 1 5 10 <210> 4 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> VL CDR1 <400> 4 Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu 1 5 10 <210> 5 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> VL CDR2 <400> 5 Val Ser Asn One <210> 6 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> VL CDR3 <400> 6 Cys Ser Gln Ser Thr His Asp Pro Tyr Thr Phe 1 5 10 <210> 7 <211> 140 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 7 Met Gly Trp Ser Tyr Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys 20 25 30 Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Ser Tyr Asn Met His Trp Val Lys Gln Thr Pro Gly Gln Gly Leu 50 55 60 Glu Trp Ile Gly Ala Ile Tyr Pro Gly Asn Gly Tyr Thr Ser Tyr Asn 65 70 75 80 Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Arg Ser Ser Ser 85 90 95 Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val 100 105 110 Tyr Phe Cys Ala Arg Trp Leu Arg Asn Tyr Ala Met Asp Tyr Trp Gly 115 120 125 Gln Gly Thr Ser Val Thr Val Ser Ser Ala Lys Thr 130 135 140 <210> 8 <211> 140 <212> PRT <213> Artificial Sequence <220> <223> VL <400> 8 Met Arg Ala Pro Ala Gln Phe Phe Gly Phe Leu Leu Leu Trp Phe Pro 1 5 10 15 Ala Ser Ser Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro 20 25 30 Val Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser 35 40 45 Leu Val His Ser Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys 50 55 60 Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe 65 70 75 80 Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 85 90 95 Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe 100 105 110 Cys Ser Gln Ser Thr His Asp Pro Tyr Thr Phe Gly Gly Gly Thr Lys 115 120 125 Leu Glu Ile Lys Arg Ala Asp Ala Ala Pro Thr Val 130 135 140 <210> 9 <211> 420 <212> DNA <213> Artificial Sequence <220> <223> VH <400> 9 atgggatgga gctatatcat cctcttcttg gtagcaacag ctacaggtgt ccactcccag 60 gtgcaactgc agcagcctgg ggctgagctg gtgaagcctg gggcctcagt gaagatgtcc 120 tgcaaggctt ctggctacac atttaccagt tacaatatgc actgggtaaa gcagacacct 180 ggacagggcc tggaatggat tggagctatt tatccaggaa atggttatac ttcctacaat 240 cagaagttca aaggcaaggc cacattgact gcagacagat cctccagcac agcctacatg 300 cagctcagca gcctgacatc tgaggactct gcggtctatt tctgtgcaag atggttacga 360 aactatgcta tggactactg gggtcaagga acctcagtca ccgtctcctc agccaaaacg 420 420 <210> 10 <211> 420 <212> DNA <213> Artificial Sequence <220> <223> VL <400> 10 atgagggccc ctgctcagtt ttttgggttc ttgttgctct ggtttccagc ttccagcagt 60 gatgttgtga tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 120 atctcttgca gatctagtca gagccttgta cacagtaatg gaaacaccta tttacattgg 180 tacctgcaga agccaggcca gtctccaaag ctcctgatct acaaagtttc caaccgattt 240 tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc 300 agcagagtgg aggctgagga tctgggagtt tatttctgct ctcaaagtac acatgatccg 360 tacacgttcg gaggggggac caagctggaa ataaaacggg ctgatgctgc accaactgta 420 420
Claims (10)
(b) 상기 (a) 단계의 배양액으로부터 제1항의 단일클론 항체 또는 이의 절편을 정제하는 단계를 포함하는, 뎅기 바이러스 4가지 혈청형의 외막 도메인 Ⅲ에 결합하는 단일클론 항체를 제조하는 방법.(a) culturing the transformant of claim 5 to produce a culture; And
(b) purifying the monoclonal antibody or the fragment thereof of claim 1 from the culture medium of step (a). 2. The method of claim 1, wherein the monoclonal antibody binds to the outer membrane domain III of the four serogroups of Dengue virus.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
KR1020160002465A KR101763745B1 (en) | 2016-01-08 | 2016-01-08 | Monoclonal antibody against envelope domain Ⅲ of four serotype dengue virus and uses thereof |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
KR1020160002465A KR101763745B1 (en) | 2016-01-08 | 2016-01-08 | Monoclonal antibody against envelope domain Ⅲ of four serotype dengue virus and uses thereof |
Publications (2)
Publication Number | Publication Date |
---|---|
KR20170085150A KR20170085150A (en) | 2017-07-24 |
KR101763745B1 true KR101763745B1 (en) | 2017-08-01 |
Family
ID=59429237
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
KR1020160002465A KR101763745B1 (en) | 2016-01-08 | 2016-01-08 | Monoclonal antibody against envelope domain Ⅲ of four serotype dengue virus and uses thereof |
Country Status (1)
Country | Link |
---|---|
KR (1) | KR101763745B1 (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR101995774B1 (en) | 2018-05-08 | 2019-07-03 | 한국세라믹기술원 | A fibronectin Extra Domain B protein scaffold specifically binding to dengue virus |
KR20200099732A (en) * | 2019-02-15 | 2020-08-25 | 원광대학교산학협력단 | Composition for early diagnosis of dengue virus infection comprising peptide derived from envelope domain Ⅲ of four serotype dengue virus as effective component and uses thereof |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR102048812B1 (en) * | 2018-12-19 | 2019-11-26 | 주식회사 엠디헬스케어 | DNA aptamer binding specifically to dengue virus envelope protein domain III(DENV EDIII) and uses thereof |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2007524350A (en) | 2003-01-31 | 2007-08-30 | ノバルティス アクチエンゲゼルシャフト | Anti-dengue virus antibodies, compositions, methods and uses |
US8637035B2 (en) | 2010-07-16 | 2014-01-28 | Academia Sinica | Anti-dengue virus antibodies |
-
2016
- 2016-01-08 KR KR1020160002465A patent/KR101763745B1/en active IP Right Grant
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2007524350A (en) | 2003-01-31 | 2007-08-30 | ノバルティス アクチエンゲゼルシャフト | Anti-dengue virus antibodies, compositions, methods and uses |
US8637035B2 (en) | 2010-07-16 | 2014-01-28 | Academia Sinica | Anti-dengue virus antibodies |
Non-Patent Citations (1)
Title |
---|
J Immunol. 2012,188(10):4971-4979.* |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR101995774B1 (en) | 2018-05-08 | 2019-07-03 | 한국세라믹기술원 | A fibronectin Extra Domain B protein scaffold specifically binding to dengue virus |
KR20200099732A (en) * | 2019-02-15 | 2020-08-25 | 원광대학교산학협력단 | Composition for early diagnosis of dengue virus infection comprising peptide derived from envelope domain Ⅲ of four serotype dengue virus as effective component and uses thereof |
KR102169928B1 (en) | 2019-02-15 | 2020-10-26 | 원광대학교산학협력단 | Composition for early diagnosis of dengue virus infection comprising peptide derived from envelope domain Ⅲ of four serotype dengue virus as effective component and uses thereof |
Also Published As
Publication number | Publication date |
---|---|
KR20170085150A (en) | 2017-07-24 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP7418508B2 (en) | Anti-pro/latent myostatin antibodies and their uses | |
EP3344654B1 (en) | Anti-lag-3 antibodies | |
US20190367589A1 (en) | Anti-jagged 1/jagged 2 cross-reactive antibodies, activatable anti-jagged antibodies and methods of use thereof | |
JP7070932B2 (en) | BAFF-R Targeted Chimeric Antigen Receptor Modified T Cells and Their Use | |
TW201731870A (en) | Human immunodeficiency virus neutralizing antibodies | |
CN109563169A (en) | Anti- HLA-G specific antibody | |
KR20140104945A (en) | Anti-axl antibodies and uses thereof | |
WO2016167306A1 (en) | ANTI-HUMAN Notch 4 ANTIBODY | |
CA2920890A1 (en) | Antibodies to plasminogen activator inhibitor-1 (pai-1) and uses thereof | |
TW201623331A (en) | Anti-MCAM antibodies and associated methods of use | |
KR20230009441A (en) | Anti-TIGIT Antibodies, Methods of Making and Uses Thereof | |
WO2022068810A1 (en) | Anti-claudin18.2 and cd3 bispecific antibody and use thereof | |
KR101763745B1 (en) | Monoclonal antibody against envelope domain Ⅲ of four serotype dengue virus and uses thereof | |
KR20200128544A (en) | PD1 binder | |
WO2021254481A9 (en) | Anti-claudin18.2 antibody and use thereof | |
KR20060097526A (en) | Monoclonal antibody specific to anthrax toxin | |
JP2021503915A (en) | Humanized antibodies targeting human tissue factor | |
KR20140043795A (en) | Soluble integrin a4 mutant | |
JP2023525156A (en) | Novel antibody that specifically binds to human CEACAM1/3/5 and use thereof | |
JP2020090528A (en) | Antibodies that cross-link human and mouse sema3a and uses thereof | |
EP4119581A1 (en) | Novel fab dimers | |
TWI816621B (en) | Antibody and application thereof | |
WO2022068809A1 (en) | Anti-cd3 antibody and uses thereof | |
KR101998029B1 (en) | Antibody specifically binding to MIC-1 protein and uses thereof | |
KR101856904B1 (en) | Antibody specifically binding to PAUF and use thereof |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AMND | Amendment | ||
AMND | Amendment | ||
X701 | Decision to grant (after re-examination) | ||
GRNT | Written decision to grant |