CN107216388A - A kind of preparation method and purposes for treating hepatitis C virus cytotoxic drug - Google Patents
A kind of preparation method and purposes for treating hepatitis C virus cytotoxic drug Download PDFInfo
- Publication number
- CN107216388A CN107216388A CN201710668064.2A CN201710668064A CN107216388A CN 107216388 A CN107216388 A CN 107216388A CN 201710668064 A CN201710668064 A CN 201710668064A CN 107216388 A CN107216388 A CN 107216388A
- Authority
- CN
- China
- Prior art keywords
- antibody
- hcv
- binding molecule
- seq
- cell
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 238000002360 preparation method Methods 0.000 title description 9
- 241000711549 Hepacivirus C Species 0.000 title description 5
- 229940127089 cytotoxic agent Drugs 0.000 title description 3
- 239000002254 cytotoxic agent Substances 0.000 title description 3
- 208000015181 infectious disease Diseases 0.000 claims abstract description 26
- 239000003814 drug Substances 0.000 claims abstract description 14
- 201000010099 disease Diseases 0.000 claims abstract description 11
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 11
- 230000027455 binding Effects 0.000 claims description 60
- 238000009739 binding Methods 0.000 claims description 55
- 238000000034 method Methods 0.000 claims description 31
- 238000001514 detection method Methods 0.000 claims description 21
- 108091033319 polynucleotide Proteins 0.000 claims description 15
- 102000040430 polynucleotide Human genes 0.000 claims description 15
- 239000002157 polynucleotide Substances 0.000 claims description 15
- 239000012634 fragment Substances 0.000 claims description 12
- 208000006454 hepatitis Diseases 0.000 claims description 9
- 238000003259 recombinant expression Methods 0.000 claims description 6
- 238000000926 separation method Methods 0.000 claims description 6
- 231100000283 hepatitis Toxicity 0.000 claims description 5
- 239000008194 pharmaceutical composition Substances 0.000 claims description 5
- 208000034189 Sclerosis Diseases 0.000 claims description 4
- 230000002440 hepatic effect Effects 0.000 claims description 4
- 206010024648 Livedo reticularis Diseases 0.000 claims description 2
- 201000010273 Porphyria Cutanea Tarda Diseases 0.000 claims description 2
- 206010036186 Porphyria non-acute Diseases 0.000 claims description 2
- 201000003278 cryoglobulinemia Diseases 0.000 claims description 2
- 201000007270 liver cancer Diseases 0.000 claims description 2
- 208000019423 liver disease Diseases 0.000 claims description 2
- 208000014018 liver neoplasm Diseases 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims 2
- 206010047115 Vasculitis Diseases 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 17
- 238000006386 neutralization reaction Methods 0.000 abstract description 11
- 230000003472 neutralizing effect Effects 0.000 abstract description 3
- 238000003745 diagnosis Methods 0.000 abstract description 2
- 230000005847 immunogenicity Effects 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 55
- 239000000427 antigen Substances 0.000 description 22
- 108091007433 antigens Proteins 0.000 description 22
- 102000036639 antigens Human genes 0.000 description 22
- 239000002773 nucleotide Substances 0.000 description 17
- 125000003729 nucleotide group Chemical group 0.000 description 17
- 108090000623 proteins and genes Proteins 0.000 description 17
- 241000700605 Viruses Species 0.000 description 15
- 230000014509 gene expression Effects 0.000 description 13
- 239000000047 product Substances 0.000 description 13
- 150000001413 amino acids Chemical group 0.000 description 11
- 239000000523 sample Substances 0.000 description 11
- 238000005516 engineering process Methods 0.000 description 10
- 102100034349 Integrase Human genes 0.000 description 9
- 239000013604 expression vector Substances 0.000 description 9
- 239000007788 liquid Substances 0.000 description 9
- 238000002965 ELISA Methods 0.000 description 8
- 101710121417 Envelope glycoprotein Proteins 0.000 description 8
- 238000006243 chemical reaction Methods 0.000 description 8
- 239000000463 material Substances 0.000 description 8
- 235000018102 proteins Nutrition 0.000 description 8
- 102000004169 proteins and genes Human genes 0.000 description 8
- 210000003719 b-lymphocyte Anatomy 0.000 description 7
- 102000039446 nucleic acids Human genes 0.000 description 7
- 108020004707 nucleic acids Proteins 0.000 description 7
- 150000007523 nucleic acids Chemical class 0.000 description 7
- 101000600434 Homo sapiens Putative uncharacterized protein encoded by MIR7-3HG Proteins 0.000 description 6
- 102100037401 Putative uncharacterized protein encoded by MIR7-3HG Human genes 0.000 description 6
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 238000001890 transfection Methods 0.000 description 6
- 241000894006 Bacteria Species 0.000 description 5
- 108020004414 DNA Proteins 0.000 description 5
- 108060001084 Luciferase Proteins 0.000 description 5
- 210000004369 blood Anatomy 0.000 description 5
- 239000008280 blood Substances 0.000 description 5
- 239000003636 conditioned culture medium Substances 0.000 description 5
- 238000010790 dilution Methods 0.000 description 5
- 239000012895 dilution Substances 0.000 description 5
- 229940079593 drug Drugs 0.000 description 5
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 5
- 230000006870 function Effects 0.000 description 5
- 239000001963 growth medium Substances 0.000 description 5
- 210000004962 mammalian cell Anatomy 0.000 description 5
- 239000013612 plasmid Substances 0.000 description 5
- 229920001184 polypeptide Polymers 0.000 description 5
- 102000004196 processed proteins & peptides Human genes 0.000 description 5
- 108090000765 processed proteins & peptides Proteins 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 4
- 108091026890 Coding region Proteins 0.000 description 4
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 4
- 230000003321 amplification Effects 0.000 description 4
- 239000002299 complementary DNA Substances 0.000 description 4
- 238000001962 electrophoresis Methods 0.000 description 4
- 238000011534 incubation Methods 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 238000003199 nucleic acid amplification method Methods 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 4
- 238000013518 transcription Methods 0.000 description 4
- 230000035897 transcription Effects 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 241000588724 Escherichia coli Species 0.000 description 3
- 208000005176 Hepatitis C Diseases 0.000 description 3
- 101000777550 Homo sapiens CCN family member 2 Proteins 0.000 description 3
- 108060003951 Immunoglobulin Proteins 0.000 description 3
- 239000005089 Luciferase Substances 0.000 description 3
- 108020004511 Recombinant DNA Proteins 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 210000001185 bone marrow Anatomy 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 229940088598 enzyme Drugs 0.000 description 3
- 102000047612 human CCN2 Human genes 0.000 description 3
- 230000001900 immune effect Effects 0.000 description 3
- 102000018358 immunoglobulin Human genes 0.000 description 3
- 210000004698 lymphocyte Anatomy 0.000 description 3
- 210000001806 memory b lymphocyte Anatomy 0.000 description 3
- 229910021645 metal ion Inorganic materials 0.000 description 3
- 239000011148 porous material Substances 0.000 description 3
- 230000002265 prevention Effects 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 238000003757 reverse transcription PCR Methods 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 238000001262 western blot Methods 0.000 description 3
- OOIBFPKQHULHSQ-UHFFFAOYSA-N (3-hydroxy-1-adamantyl) 2-methylprop-2-enoate Chemical compound C1C(C2)CC3CC2(O)CC1(OC(=O)C(=C)C)C3 OOIBFPKQHULHSQ-UHFFFAOYSA-N 0.000 description 2
- RZVAJINKPMORJF-UHFFFAOYSA-N Acetaminophen Chemical compound CC(=O)NC1=CC=C(O)C=C1 RZVAJINKPMORJF-UHFFFAOYSA-N 0.000 description 2
- QOIGKCBMXUCDQU-KDXUFGMBSA-N Ala-Thr-Pro Chemical compound C[C@H]([C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](C)N)O QOIGKCBMXUCDQU-KDXUFGMBSA-N 0.000 description 2
- 102100022005 B-lymphocyte antigen CD20 Human genes 0.000 description 2
- 235000014469 Bacillus subtilis Nutrition 0.000 description 2
- 241000283707 Capra Species 0.000 description 2
- 241000701489 Cauliflower mosaic virus Species 0.000 description 2
- 206010008909 Chronic Hepatitis Diseases 0.000 description 2
- XTQFHTHIAKKCTM-YFKPBYRVSA-N Gly-Glu-Gly Chemical compound NCC(=O)N[C@@H](CCC(O)=O)C(=O)NCC(O)=O XTQFHTHIAKKCTM-YFKPBYRVSA-N 0.000 description 2
- YWAQATDNEKZFFK-BYPYZUCNSA-N Gly-Gly-Ser Chemical compound NCC(=O)NCC(=O)N[C@@H](CO)C(O)=O YWAQATDNEKZFFK-BYPYZUCNSA-N 0.000 description 2
- OJNZVYSGVYLQIN-BQBZGAKWSA-N Gly-Met-Asp Chemical compound [H]NCC(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(O)=O OJNZVYSGVYLQIN-BQBZGAKWSA-N 0.000 description 2
- 101000897405 Homo sapiens B-lymphocyte antigen CD20 Proteins 0.000 description 2
- PDIDTSZKKFEDMB-UWVGGRQHSA-N Lys-Pro-Gly Chemical compound [H]N[C@@H](CCCCN)C(=O)N1CCC[C@H]1C(=O)NCC(O)=O PDIDTSZKKFEDMB-UWVGGRQHSA-N 0.000 description 2
- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 description 2
- BSKMOCNNLNDIMU-CDMKHQONSA-N Phe-Thr-Gly Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(O)=O BSKMOCNNLNDIMU-CDMKHQONSA-N 0.000 description 2
- FRMKIPSIZSFTTE-HJOGWXRNSA-N Phe-Tyr-Phe Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O FRMKIPSIZSFTTE-HJOGWXRNSA-N 0.000 description 2
- 241000235648 Pichia Species 0.000 description 2
- 239000004697 Polyetherimide Substances 0.000 description 2
- 241000235070 Saccharomyces Species 0.000 description 2
- GXUWHVZYDAHFSV-FLBSBUHZSA-N Thr-Ile-Thr Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(O)=O GXUWHVZYDAHFSV-FLBSBUHZSA-N 0.000 description 2
- ZESGVALRVJIVLZ-VFCFLDTKSA-N Thr-Thr-Pro Chemical compound C[C@H]([C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)N1CCC[C@@H]1C(=O)O)N)O ZESGVALRVJIVLZ-VFCFLDTKSA-N 0.000 description 2
- 241000218636 Thuja Species 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 230000001154 acute effect Effects 0.000 description 2
- 239000012491 analyte Substances 0.000 description 2
- 230000010056 antibody-dependent cellular cytotoxicity Effects 0.000 description 2
- 230000033228 biological regulation Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 229960002685 biotin Drugs 0.000 description 2
- 235000020958 biotin Nutrition 0.000 description 2
- 239000011616 biotin Substances 0.000 description 2
- 210000001124 body fluid Anatomy 0.000 description 2
- 239000010839 body fluid Substances 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 230000010261 cell growth Effects 0.000 description 2
- 230000004087 circulation Effects 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 230000000295 complement effect Effects 0.000 description 2
- 238000005336 cracking Methods 0.000 description 2
- 230000007850 degeneration Effects 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 239000003085 diluting agent Substances 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000001215 fluorescent labelling Methods 0.000 description 2
- VPZXBVLAVMBEQI-UHFFFAOYSA-N glycyl-DL-alpha-alanine Natural products OC(=O)C(C)NC(=O)CN VPZXBVLAVMBEQI-UHFFFAOYSA-N 0.000 description 2
- 108010074027 glycyl-seryl-phenylalanine Proteins 0.000 description 2
- 108010089804 glycyl-threonine Proteins 0.000 description 2
- 230000016784 immunoglobulin production Effects 0.000 description 2
- 210000000554 iris Anatomy 0.000 description 2
- 239000006166 lysate Substances 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000002703 mutagenesis Methods 0.000 description 2
- 231100000350 mutagenesis Toxicity 0.000 description 2
- 229940005654 nitrite ion Drugs 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 239000000825 pharmaceutical preparation Substances 0.000 description 2
- 229920001601 polyetherimide Polymers 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 108010029020 prolylglycine Proteins 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000000197 pyrolysis Methods 0.000 description 2
- 239000012857 radioactive material Substances 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000009870 specific binding Effects 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 238000002560 therapeutic procedure Methods 0.000 description 2
- 231100000167 toxic agent Toxicity 0.000 description 2
- 239000003440 toxic substance Substances 0.000 description 2
- 229940072651 tylenol Drugs 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
- DSSYKIVIOFKYAU-XCBNKYQSSA-N (R)-camphor Chemical compound C1C[C@@]2(C)C(=O)C[C@@H]1C2(C)C DSSYKIVIOFKYAU-XCBNKYQSSA-N 0.000 description 1
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- PXFBZOLANLWPMH-UHFFFAOYSA-N 16-Epiaffinine Natural products C1C(C2=CC=CC=C2N2)=C2C(=O)CC2C(=CC)CN(C)C1C2CO PXFBZOLANLWPMH-UHFFFAOYSA-N 0.000 description 1
- OALHHIHQOFIMEF-UHFFFAOYSA-N 3',6'-dihydroxy-2',4',5',7'-tetraiodo-3h-spiro[2-benzofuran-1,9'-xanthene]-3-one Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC(I)=C(O)C(I)=C1OC1=C(I)C(O)=C(I)C=C21 OALHHIHQOFIMEF-UHFFFAOYSA-N 0.000 description 1
- CJIJXIFQYOPWTF-UHFFFAOYSA-N 7-hydroxycoumarin Natural products O1C(=O)C=CC2=CC(O)=CC=C21 CJIJXIFQYOPWTF-UHFFFAOYSA-N 0.000 description 1
- 102000012440 Acetylcholinesterase Human genes 0.000 description 1
- 108010022752 Acetylcholinesterase Proteins 0.000 description 1
- 206010065051 Acute hepatitis C Diseases 0.000 description 1
- 108010000239 Aequorin Proteins 0.000 description 1
- WQKAQKZRDIZYNV-VZFHVOOUSA-N Ala-Ser-Thr Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(O)=O WQKAQKZRDIZYNV-VZFHVOOUSA-N 0.000 description 1
- ARHJJAAWNWOACN-FXQIFTODSA-N Ala-Ser-Val Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(O)=O ARHJJAAWNWOACN-FXQIFTODSA-N 0.000 description 1
- XQNRANMFRPCFFW-GCJQMDKQSA-N Ala-Thr-Asn Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](C)N)O XQNRANMFRPCFFW-GCJQMDKQSA-N 0.000 description 1
- CREYEAPXISDKSB-FQPOAREZSA-N Ala-Thr-Tyr Chemical compound [H]N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O CREYEAPXISDKSB-FQPOAREZSA-N 0.000 description 1
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- HQIZDMIGUJOSNI-IUCAKERBSA-N Arg-Gly-Arg Chemical compound N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(O)=O HQIZDMIGUJOSNI-IUCAKERBSA-N 0.000 description 1
- LXMKTIZAGIBQRX-HRCADAONSA-N Arg-Phe-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC2=CC=CC=C2)NC(=O)[C@H](CCCN=C(N)N)N)C(=O)O LXMKTIZAGIBQRX-HRCADAONSA-N 0.000 description 1
- AOHKLEBWKMKITA-IHRRRGAJSA-N Arg-Phe-Ser Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CO)C(=O)O)NC(=O)[C@H](CCCN=C(N)N)N AOHKLEBWKMKITA-IHRRRGAJSA-N 0.000 description 1
- GQRDIVQPSMPQME-ZPFDUUQYSA-N Asn-Ile-Leu Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(C)C)C(O)=O GQRDIVQPSMPQME-ZPFDUUQYSA-N 0.000 description 1
- HFPXZWPUVFVNLL-GUBZILKMSA-N Asn-Leu-Gln Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O HFPXZWPUVFVNLL-GUBZILKMSA-N 0.000 description 1
- JTXVXGXTRXMOFJ-FXQIFTODSA-N Asn-Pro-Asn Chemical compound NC(=O)C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(N)=O)C(O)=O JTXVXGXTRXMOFJ-FXQIFTODSA-N 0.000 description 1
- HPBNLFLSSQDFQW-WHFBIAKZSA-N Asn-Ser-Gly Chemical compound NC(=O)C[C@H](N)C(=O)N[C@@H](CO)C(=O)NCC(O)=O HPBNLFLSSQDFQW-WHFBIAKZSA-N 0.000 description 1
- WLVLIYYBPPONRJ-GCJQMDKQSA-N Asn-Thr-Ala Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(O)=O WLVLIYYBPPONRJ-GCJQMDKQSA-N 0.000 description 1
- XYBJLTKSGFBLCS-QXEWZRGKSA-N Asp-Arg-Val Chemical compound NC(N)=NCCC[C@@H](C(=O)N[C@@H](C(C)C)C(O)=O)NC(=O)[C@@H](N)CC(O)=O XYBJLTKSGFBLCS-QXEWZRGKSA-N 0.000 description 1
- KYQNAIMCTRZLNP-QSFUFRPTSA-N Asp-Ile-Val Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C(C)C)C(O)=O KYQNAIMCTRZLNP-QSFUFRPTSA-N 0.000 description 1
- MNQMTYSEKZHIDF-GCJQMDKQSA-N Asp-Thr-Ala Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(O)=O MNQMTYSEKZHIDF-GCJQMDKQSA-N 0.000 description 1
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- 108090001008 Avidin Proteins 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- LSNNMFCWUKXFEE-UHFFFAOYSA-M Bisulfite Chemical compound OS([O-])=O LSNNMFCWUKXFEE-UHFFFAOYSA-M 0.000 description 1
- 208000006154 Chronic hepatitis C Diseases 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- SZQCDCKIGWQAQN-FXQIFTODSA-N Cys-Arg-Ala Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(O)=O SZQCDCKIGWQAQN-FXQIFTODSA-N 0.000 description 1
- ULGZDMOVFRHVEP-RWJQBGPGSA-N Erythromycin Chemical compound O([C@@H]1[C@@H](C)C(=O)O[C@@H]([C@@]([C@H](O)[C@@H](C)C(=O)[C@H](C)C[C@@](C)(O)[C@H](O[C@H]2[C@@H]([C@H](C[C@@H](C)O2)N(C)C)O)[C@H]1C)(C)O)CC)[C@H]1C[C@@](C)(OC)[C@@H](O)[C@H](C)O1 ULGZDMOVFRHVEP-RWJQBGPGSA-N 0.000 description 1
- OYTPNWYZORARHL-XHNCKOQMSA-N Gln-Ala-Pro Chemical compound C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CCC(=O)N)N OYTPNWYZORARHL-XHNCKOQMSA-N 0.000 description 1
- VSXBYIJUAXPAAL-WDSKDSINSA-N Gln-Gly-Ala Chemical compound OC(=O)[C@H](C)NC(=O)CNC(=O)[C@@H](N)CCC(N)=O VSXBYIJUAXPAAL-WDSKDSINSA-N 0.000 description 1
- KQOPMGBHNQBCEL-HVTMNAMFSA-N Gln-His-Ile Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O KQOPMGBHNQBCEL-HVTMNAMFSA-N 0.000 description 1
- OSCLNNWLKKIQJM-WDSKDSINSA-N Gln-Ser-Gly Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)NCC(O)=O OSCLNNWLKKIQJM-WDSKDSINSA-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
- PAQUJCSYVIBPLC-AVGNSLFASA-N Glu-Asp-Phe Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 PAQUJCSYVIBPLC-AVGNSLFASA-N 0.000 description 1
- LVCHEMOPBORRLB-DCAQKATOSA-N Glu-Gln-Lys Chemical compound NCCCC[C@H](NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](N)CCC(O)=O)C(O)=O LVCHEMOPBORRLB-DCAQKATOSA-N 0.000 description 1
- MWMJCGBSIORNCD-AVGNSLFASA-N Glu-Leu-Leu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O MWMJCGBSIORNCD-AVGNSLFASA-N 0.000 description 1
- ZYRXTRTUCAVNBQ-GVXVVHGQSA-N Glu-Val-Lys Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CCC(=O)O)N ZYRXTRTUCAVNBQ-GVXVVHGQSA-N 0.000 description 1
- LEGMTEAZGRRIMY-ZKWXMUAHSA-N Gly-Cys-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CS)NC(=O)CN LEGMTEAZGRRIMY-ZKWXMUAHSA-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
- TVTZEOHWHUVYCG-KYNKHSRBSA-N Gly-Thr-Thr Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O TVTZEOHWHUVYCG-KYNKHSRBSA-N 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- QMUHTRISZMFKAY-MXAVVETBSA-N His-Ile-Lys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CC1=CN=CN1)N QMUHTRISZMFKAY-MXAVVETBSA-N 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- 101100321817 Human parvovirus B19 (strain HV) 7.5K gene Proteins 0.000 description 1
- UKTUOMWSJPXODT-GUDRVLHUSA-N Ile-Asn-Pro Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N1CCC[C@@H]1C(=O)O)N UKTUOMWSJPXODT-GUDRVLHUSA-N 0.000 description 1
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 1
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 1
- 108700001097 Insect Genes Proteins 0.000 description 1
- 102000014150 Interferons Human genes 0.000 description 1
- 108010050904 Interferons Proteins 0.000 description 1
- PMGDADKJMCOXHX-UHFFFAOYSA-N L-Arginyl-L-glutamin-acetat Natural products NC(=N)NCCCC(N)C(=O)NC(CCC(N)=O)C(O)=O PMGDADKJMCOXHX-UHFFFAOYSA-N 0.000 description 1
- CQGSYZCULZMEDE-UHFFFAOYSA-N Leu-Gln-Pro Natural products CC(C)CC(N)C(=O)NC(CCC(N)=O)C(=O)N1CCCC1C(O)=O CQGSYZCULZMEDE-UHFFFAOYSA-N 0.000 description 1
- MVJRBCJCRYGCKV-GVXVVHGQSA-N Leu-Val-Gln Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O MVJRBCJCRYGCKV-GVXVVHGQSA-N 0.000 description 1
- IXHKPDJKKCUKHS-GARJFASQSA-N Lys-Ala-Pro Chemical compound C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CCCCN)N IXHKPDJKKCUKHS-GARJFASQSA-N 0.000 description 1
- WALVCOOOKULCQM-ULQDDVLXSA-N Lys-Arg-Phe Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O WALVCOOOKULCQM-ULQDDVLXSA-N 0.000 description 1
- YTJFXEDRUOQGSP-DCAQKATOSA-N Lys-Pro-Ser Chemical compound [H]N[C@@H](CCCCN)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(O)=O YTJFXEDRUOQGSP-DCAQKATOSA-N 0.000 description 1
- QLFAPXUXEBAWEK-NHCYSSNCSA-N Lys-Val-Asp Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O QLFAPXUXEBAWEK-NHCYSSNCSA-N 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- OOSPRDCGTLQLBP-NHCYSSNCSA-N Met-Glu-Val Chemical compound [H]N[C@@H](CCSC)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O OOSPRDCGTLQLBP-NHCYSSNCSA-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
- 101710135898 Myc proto-oncogene protein Proteins 0.000 description 1
- 102100038895 Myc proto-oncogene protein Human genes 0.000 description 1
- KZNQNBZMBZJQJO-UHFFFAOYSA-N N-glycyl-L-proline Natural products NCC(=O)N1CCCC1C(O)=O KZNQNBZMBZJQJO-UHFFFAOYSA-N 0.000 description 1
- 108010079364 N-glycylalanine Proteins 0.000 description 1
- 108010002311 N-glycylglutamic acid Proteins 0.000 description 1
- 108091007491 NSP3 Papain-like protease domains Proteins 0.000 description 1
- 244000061176 Nicotiana tabacum Species 0.000 description 1
- 235000002637 Nicotiana tabacum Nutrition 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 108700022034 Opsonin Proteins Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- KLYYKKGCPOGDPE-OEAJRASXSA-N Phe-Thr-Leu Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(O)=O KLYYKKGCPOGDPE-OEAJRASXSA-N 0.000 description 1
- 108010004729 Phycoerythrin Proteins 0.000 description 1
- ZTVCLZLGHZXLOT-ULQDDVLXSA-N Pro-Glu-Trp Chemical compound C1C[C@H](NC1)C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CC2=CNC3=CC=CC=C32)C(=O)O ZTVCLZLGHZXLOT-ULQDDVLXSA-N 0.000 description 1
- VTFXTWDFPTWNJY-RHYQMDGZSA-N Pro-Leu-Thr Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O VTFXTWDFPTWNJY-RHYQMDGZSA-N 0.000 description 1
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 1
- 241000711798 Rabies lyssavirus Species 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 241000235342 Saccharomycetes Species 0.000 description 1
- UQFYNFTYDHUIMI-WHFBIAKZSA-N Ser-Gly-Ala Chemical compound OC(=O)[C@H](C)NC(=O)CNC(=O)[C@@H](N)CO UQFYNFTYDHUIMI-WHFBIAKZSA-N 0.000 description 1
- MIJWOJAXARLEHA-WDSKDSINSA-N Ser-Gly-Glu Chemical compound OC[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCC(O)=O MIJWOJAXARLEHA-WDSKDSINSA-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
- DINQYZRMXGWWTG-GUBZILKMSA-N Ser-Pro-Pro Chemical compound OC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N1[C@H](C(O)=O)CCC1 DINQYZRMXGWWTG-GUBZILKMSA-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
- OJFFAQFRCVPHNN-JYBASQMISA-N Ser-Thr-Trp Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O OJFFAQFRCVPHNN-JYBASQMISA-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
- JGUWRQWULDWNCM-FXQIFTODSA-N Ser-Val-Ser Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(O)=O JGUWRQWULDWNCM-FXQIFTODSA-N 0.000 description 1
- 108010003723 Single-Domain Antibodies Proteins 0.000 description 1
- 108010090804 Streptavidin Proteins 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 239000005864 Sulphur Substances 0.000 description 1
- GLQFKOVWXPPFTP-VEVYYDQMSA-N Thr-Arg-Asp Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(O)=O GLQFKOVWXPPFTP-VEVYYDQMSA-N 0.000 description 1
- JVTHIXKSVYEWNI-JRQIVUDYSA-N Thr-Asn-Tyr Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O JVTHIXKSVYEWNI-JRQIVUDYSA-N 0.000 description 1
- UHBPFYOQQPFKQR-JHEQGTHGSA-N Thr-Gln-Gly Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(N)=O)C(=O)NCC(O)=O UHBPFYOQQPFKQR-JHEQGTHGSA-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
- IVDFVBVIVLJJHR-LKXGYXEUSA-N Thr-Ser-Asp Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(O)=O IVDFVBVIVLJJHR-LKXGYXEUSA-N 0.000 description 1
- NQQMWWVVGIXUOX-SVSWQMSJSA-N Thr-Ser-Ile Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O NQQMWWVVGIXUOX-SVSWQMSJSA-N 0.000 description 1
- 101710150448 Transcriptional regulator Myc Proteins 0.000 description 1
- SVGAWGVHFIYAEE-JSGCOSHPSA-N Trp-Gly-Gln Chemical compound C1=CC=C2C(C[C@H](N)C(=O)NCC(=O)N[C@@H](CCC(N)=O)C(O)=O)=CNC2=C1 SVGAWGVHFIYAEE-JSGCOSHPSA-N 0.000 description 1
- MBLJBGZWLHTJBH-SZMVWBNQSA-N Trp-Val-Arg Chemical compound C1=CC=C2C(C[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O)=CNC2=C1 MBLJBGZWLHTJBH-SZMVWBNQSA-N 0.000 description 1
- NOXKHHXSHQFSGJ-FQPOAREZSA-N Tyr-Ala-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 NOXKHHXSHQFSGJ-FQPOAREZSA-N 0.000 description 1
- QOIKZODVIPOPDD-AVGNSLFASA-N Tyr-Cys-Gln Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(O)=O QOIKZODVIPOPDD-AVGNSLFASA-N 0.000 description 1
- QUILOGWWLXMSAT-IHRRRGAJSA-N Tyr-Gln-Gln Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O QUILOGWWLXMSAT-IHRRRGAJSA-N 0.000 description 1
- DZKFGCNKEVMXFA-JUKXBJQTSA-N Tyr-Ile-His Chemical compound CC[C@H](C)[C@H](NC(=O)[C@@H](N)Cc1ccc(O)cc1)C(=O)N[C@@H](Cc1cnc[nH]1)C(O)=O DZKFGCNKEVMXFA-JUKXBJQTSA-N 0.000 description 1
- 241000700618 Vaccinia virus Species 0.000 description 1
- VJOWWOGRNXRQMF-UVBJJODRSA-N Val-Ala-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@H](C)NC(=O)[C@@H](N)C(C)C)C(O)=O)=CNC2=C1 VJOWWOGRNXRQMF-UVBJJODRSA-N 0.000 description 1
- SSYBNWFXCFNRFN-GUBZILKMSA-N Val-Pro-Ser Chemical compound CC(C)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(O)=O SSYBNWFXCFNRFN-GUBZILKMSA-N 0.000 description 1
- JQTYTBPCSOAZHI-FXQIFTODSA-N Val-Ser-Cys Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CS)C(=O)O)N JQTYTBPCSOAZHI-FXQIFTODSA-N 0.000 description 1
- LCHZBEUVGAVMKS-RHYQMDGZSA-N Val-Thr-Leu Chemical compound CC(C)C[C@H](NC(=O)[C@@H](NC(=O)[C@@H](N)C(C)C)[C@@H](C)O)C(O)=O LCHZBEUVGAVMKS-RHYQMDGZSA-N 0.000 description 1
- HTONZBWRYUKUKC-RCWTZXSCSA-N Val-Thr-Val Chemical compound CC(C)[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(O)=O HTONZBWRYUKUKC-RCWTZXSCSA-N 0.000 description 1
- OWFGFHQMSBTKLX-UFYCRDLUSA-N Val-Tyr-Tyr Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CC2=CC=C(C=C2)O)C(=O)O)N OWFGFHQMSBTKLX-UFYCRDLUSA-N 0.000 description 1
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 1
- 229940022698 acetylcholinesterase Drugs 0.000 description 1
- 208000037621 acute hepatitis C virus infection Diseases 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 108010069020 alanyl-prolyl-glycine Proteins 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 230000010100 anticoagulation Effects 0.000 description 1
- 108010008355 arginyl-glutamine Proteins 0.000 description 1
- 108010040443 aspartyl-aspartic acid Proteins 0.000 description 1
- 108010069205 aspartyl-phenylalanine Proteins 0.000 description 1
- 102000005936 beta-Galactosidase Human genes 0.000 description 1
- 108010005774 beta-Galactosidase Proteins 0.000 description 1
- 238000001574 biopsy Methods 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 229960000846 camphor Drugs 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000001311 chemical methods and process Methods 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 1
- 239000013599 cloning vector Substances 0.000 description 1
- 238000004891 communication Methods 0.000 description 1
- 230000004540 complement-dependent cytotoxicity Effects 0.000 description 1
- 230000008473 connective tissue growth Effects 0.000 description 1
- 108091036078 conserved sequence Proteins 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- 239000000890 drug combination Substances 0.000 description 1
- 229940126534 drug product Drugs 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 108010078144 glutaminyl-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
- 108010015792 glycyllysine Proteins 0.000 description 1
- 108010037850 glycylvaline 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
- 230000012010 growth Effects 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 208000010710 hepatitis C virus infection Diseases 0.000 description 1
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 1
- 231100000844 hepatocellular carcinoma Toxicity 0.000 description 1
- 241000411851 herbal medicine Species 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 210000004408 hybridoma Anatomy 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 230000036737 immune function Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 229940127121 immunoconjugate Drugs 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 229940079322 interferon Drugs 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- HWYHZTIRURJOHG-UHFFFAOYSA-N luminol Chemical compound O=C1NNC(=O)C2=C1C(N)=CC=C2 HWYHZTIRURJOHG-UHFFFAOYSA-N 0.000 description 1
- 108010038320 lysylphenylalanine Proteins 0.000 description 1
- 238000007726 management method Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- 239000003607 modifier Substances 0.000 description 1
- 229940125645 monoclonal antibody drug Drugs 0.000 description 1
- 229940126619 mouse monoclonal antibody Drugs 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- ZTLGJPIZUOVDMT-UHFFFAOYSA-N n,n-dichlorotriazin-4-amine Chemical compound ClN(Cl)C1=CC=NN=N1 ZTLGJPIZUOVDMT-UHFFFAOYSA-N 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 239000002777 nucleoside Substances 0.000 description 1
- 150000003833 nucleoside derivatives Chemical class 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 230000005298 paramagnetic effect Effects 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 108010070409 phenylalanyl-glycyl-glycine Proteins 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 108010031719 prolyl-serine Proteins 0.000 description 1
- 108010090894 prolylleucine Proteins 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 238000002741 site-directed mutagenesis Methods 0.000 description 1
- 229940126586 small molecule drug Drugs 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 238000012358 sourcing Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000003325 tomography Methods 0.000 description 1
- 238000003151 transfection method Methods 0.000 description 1
- 239000012096 transfection reagent Substances 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 108010044292 tryptophyltyrosine Proteins 0.000 description 1
- 108010003137 tyrosyltyrosine Proteins 0.000 description 1
- ORHBXUUXSCNDEV-UHFFFAOYSA-N umbelliferone Chemical compound C1=CC(=O)OC2=CC(O)=CC=C21 ORHBXUUXSCNDEV-UHFFFAOYSA-N 0.000 description 1
- HFTAFOQKODTIJY-UHFFFAOYSA-N umbelliferone Natural products Cc1cc2C=CC(=O)Oc2cc1OCC=CC(C)(C)O HFTAFOQKODTIJY-UHFFFAOYSA-N 0.000 description 1
- 241000701161 unidentified adenovirus Species 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
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
- C07K16/1081—Togaviridae, e.g. flavivirus, rubella virus, hog cholera virus
- C07K16/109—Hepatitis C virus; Hepatitis G virus
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/18—Togaviridae; Flaviviridae
- G01N2333/183—Flaviviridae, e.g. pestivirus, mucosal disease virus, bovine viral diarrhoea virus, classical swine fever virus (hog cholera virus) or border disease virus
- G01N2333/186—Hepatitis C; Hepatitis NANB
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/10—Detection of antigens from microorganism in sample from host
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Virology (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- Hematology (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Urology & Nephrology (AREA)
- Organic Chemistry (AREA)
- Food Science & Technology (AREA)
- Pathology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- General Physics & Mathematics (AREA)
- Microbiology (AREA)
- Communicable Diseases (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Tropical Medicine & Parasitology (AREA)
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a kind of monoclonal neutralizing antibody of full people source HCV-Ab IgG.Checking shows that the antibody can be specifically bound with HCV, therefore the antibody of the present invention can be used for the product for preparing diagnosis HCV.In addition, the antibody of the present invention has preferable HCV neutralization activities, and then HCV infection can be prevented to cause relevant disease, and the antibody of the present invention is human antibody, there is the antibody compared with mouse source, the chimeric and lower immunogenicity of humanized antibody, therefore the present invention can be used for preparing the medicine for treating or preventing HCV relevant diseases for it.
Description
Technical field
The invention belongs to cellular immunology, biology field, it is related to a kind of monoclonal antibody drug in full people source, especially
It is related to a kind of monoclonal neutralizing antibody medicine of full people source anti-hepatitis c virus.In addition, anti-the invention further relates to the neutralization
The preparation method and purposes of body medicine.
Background technology
Hepatitis C is caused by HCV (HCV) infects, mainly by blood/body fluid communication.According to world health group
Estimation is knitted, the whole world there are 1.7 hundred million people's HCV infections.It is 0.7%~3.1%, about 38,000,000 in China's healthy population HCV-Ab IgG positive rate
People.Due to many factors such as viral biology feature and host immune functions, immunity of organism is often difficult to effectively remove virus,
About 50%~80%HCV the infecteds are caused to develop into chronic hepatitis, wherein 20%~30% will develop into hepatic sclerosis.Hepatic sclerosis is suffered from
There is 1%~4% to develop into hepatocellular carcinoma every year in person.
At present, the medicine for hepatitis C have interferon, nucleoside analog, polypeptide drug and Chinese herbal medicine, with
And small-molecule drug etc., though every kind of medicine is with different degrees of curative effect, fail thoroughly to eradicate HCV.With the hair of research
Exhibition, therapeutic antibodies become the new direction for the treatment of hepatitis c.
Therapeutic antibodies are the more aggressive crude drug products of growth in recent years, to so far, about 30 kinds of Dan Ke
The granted diagnosis and treatment listed for a variety of diseases of grand antibody.In the treatment and research of various acute and chronic hepatitis,
The application of monoclonal antibody is particularly extensively and with irreplaceable effect.For development process, monoclonal antibody can be divided into
The antibody of mouse monoclonal antibody, mouse source antibody humanization and complete three kinds of stage types of humanized's monoclonal antibody.Though mouse monoclonal resists
So have that acquisition methods are more ripe, and antibody has the advantages such as high-affinity, but be accompanied by the interior antibody and partly decline in vivo
Phase is short, the anti-mouse rejection of people (human anti-murine-antibody response, HAMA) strong, antibody-dependant
The cell toxicant of cell toxicant (antibody-dependent cell cytotoxicity, ADCC) and Complement Dependent
(complement-dependent cytotoxicity, CDC) acts on the appearance of the improper drawback of excitation intensity, so as to limit
Effective application of this antibody-like.Mouse source antibody humanization is that chimeric antibody is by the sequence of the partial sequence displacement adult of mouse antibody
Row, this method reduces HAMA to a certain extent, but affinity is also decreased.In order to obtain safer, efficient Dan Ke
Grand antibody, researcher has attempted a variety of monoclonal antibody methodologies for obtaining full humanized, and relatively conventional has, and utilizes gene work
Journey mouse prepares monoclonal antibody, phage antibody library technique and single bone-marrow-derived lymphocyte antibody production techniques etc..
Single bone-marrow-derived lymphocyte antibody production techniques involved in the present invention, are that unicellular separation identification technology combination is more
A kind of vitro expression systems of monoclonal antibody of round pcr formation are planted, i.e., full people source is directly obtained from human B lymphocyte
The method of monoclonal antibody.The antibody of generation has the advantages such as full humanized, height antigentic specificity, compatibility, swollen in treatment
The advantage and good application prospect of uniqueness are showed in terms of knurl, infectious diseases, autoimmune disease and organ transplant.
Therefore, it is highly desirable to the advanced efficient monoclonal antibody technology of preparing of selection and prepares Antybody therapy and prevention HCV infection.
The content of the invention
It is an object of the invention to provide a kind of binding molecule with HCV (HCV), the binding molecule is to third
Hepatitis virus has effective neutralization.
To achieve these goals, present invention employs following technical scheme：
The invention provides a kind of binding molecule of separation, the binding molecule includes：
(1)SEQ ID NO:Heavy chain CDR1, SEQ ID NO shown in 2:Heavy chain CDR2, SEQ ID NO shown in 3:4 institutes
The heavy chain CDR3 shown；And/or
(2)SEQ ID NO:Light chain CDR1, SEQ ID NO shown in 6:Light chain CDR2, SEQ ID NO shown in 7:8 institutes
The light chain CDR3 shown.
As one aspect of the present invention, binding molecule of the invention includes：
(1) weight chain variable district, the weight chain variable district has SEQ ID NO:Amino acid sequence shown in 1；And/or
(2) light chain variable district, the light chain variable district has SEQ ID NO:Amino acid sequence shown in 5.
" binding molecule " of the present invention refers to antibody molecule and the fragment with immunologic competence, that is, contains immunologic opsonin
With reference to the molecule of the antigen binding site of antigen.The present invention antibody molecule can be any types (such as IgG, IgE, IgM,
IgD, IgA), the antibody molecule of any classification (such as IgG1, IgG2, IgG3, IgG4, IgA1, hIgA2) or subclass.Antibody point
The immunologic competence part of son includes but is not limited to Fab, Fab ' and F (ab ') 2, Fd, scFv (scFv), single-chain antibody, two sulphur
Fv (sdFv) and include the single domain antibody of VL or VH domains that key is connected.Combination antigen including single-chain antibody it is anti-
Body fragment can include single variable region or with all or part of following variable region combined：Hinge region, CH1, CH2
With CH3 domains.The present invention also includes antigen-binding fragment, and it includes variable region and hinge region, CH1, CH2 and CH3 domain
Any combination.
The binding molecule of the present invention also includes recombinating with polypeptide merging or chemically conjugated (including covalent and non-covalent sew
Close) binding molecule.
The example of polypeptide restructuring fusion includes merging the binding molecule of the present invention with label sequence, label sequence bag
Include but be not limited to HA labels, 6xHis labels, c-Myc labels, flag labels.
Chemically conjugated example includes the binding molecule of the present invention being conjugated with detectable substance.By the way that antibody coupling is arrived
Detectable substance can promote detection.The example of detectable substance includes various enzymes, prothetic group, fluorescent material, luminescent material, biology
Luminescent material, radioactive material, the positron emitting metal using various positron emission computerized tomographies and on-radiation
Paramagnetic metal ion.Using techniques known in the art, detectable substance directly can be coupled with antibody (or its fragment)
Or it is conjugated, or be coupled indirectly or conjugated through intermediate (such as joint known in the art).See, for example, on metal ion
United States Patent (USP) No.4,741,900, wherein the metal ion can be with antibody conjugate, the diagnosticum as the present invention.Properly
Enzyme example include horseradish peroxidase, alkaline phosphatase, beta galactosidase or acetylcholinesterase；It is suitable auxiliary
The example of base complex includes streptavidin/biotin and avidin/biotin；Suitable fluorescent material
Example include umbelliferone, fluorescein, fluorescein isothiocynate, rhodamine, dichlorotriazinylamine fluoresceins, pellet
Sulfonic acid chloride or phycoerythrin；The example of luminescent material includes luminol；The example of bioluminescent material includes luciferase, fluorescent
Element and aequorin；The example of suitable radioactive material includes125I、131I、111In or99Tc。
The binding molecule of the present invention also includes the functional variety of foregoing binding molecule.If becoming physical efficiency and parental generation knot
Close molecule competition specific binding HCV or its protein fragments, then it is assumed that the Variant molecules are binding molecules of the present invention
Functional variety.In other words, the functional variety remains to combine HCV or its protein fragments.The functional variety
Can have conserved sequence modification, including 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, addition and missing.These modifications can oneself knows by this area mark
The mutagenesis of quasi- technological sourcing, such as directed mutagenesis and random PCR mediation, and natural and alpha-non-natural amino acid can be included.This
Outside, functional variety can include truncate of the amino acid sequence at amino terminal or carboxyl terminal or this two ends.The present invention's
Functional variety can have identical or different, higher or lower binding affinity compared with parent binding molecule, but remain to
With reference to HCV or its fragment.The functional variety of the present invention has neutralization activity for HCV.In described
Can be with identical or higher or lower compared with parent binding molecule with activity.Hereafter, when using term " binding molecule ", its
It is also covered by the functional variety of the binding molecule.
As another aspect of the present invention, present invention also offers the polynucleotides of foregoing binding molecule.This hair
It is bright be additionally included in it is strict or it is relatively low it is strict under conditions of can be with the polynucleotides hybridization of the binding molecule of the coding present invention multinuclear
Thuja acid.
It will be appreciated by persons skilled in the art that the functional variety of these polynucleotides is also the part of the present invention.Function becomes
Body is such nucleotide sequence, can directly be translated it to provide with dividing from parent nucleic by using standard genetic code
The sequence identical amino acid sequence translated in son.
Polynucleotide sequence can be obtained using any known method in this area, and determine the nucleotides of polynucleotides
Sequence.For example, if it is known that the nucleotide sequence of antibody, it is possible to which it is described anti-that the oligonucleotides chemically synthesized assembles coding
The polynucleotides of body.
Or, the polynucleotides of encoding antibody can be produced from from the nucleic acid of suitable source.If can not be contained
There are the clone of the nucleic acid of encoding particular antibodies, but the sequence of the known antibody molecule, then can be obtained by chemical synthesis
Encode the nucleic acid of the immunoglobulin, or using the interfertile synthetic primer in 3 ' and 5 ' ends with the sequence from suitable
Source (such as antibody cDNA library or for example selected for expressing this from any tissue or cell for expressing the antibody
CDNA library produced by the hybridoma of the antibody of invention, or the therefrom separated nucleic acid arrived, preferably poly- A+RNA) it is logical
The nucleic acid that PCR amplifications obtain encoding the immunoglobulin is crossed, or by using the few core for being specific to the specific gene sequence
Thuja acid probe obtains encoding the nucleic acid of the immunoglobulin by clone, described for example to identify coding from cDNA library
The cDNA clone of antibody.Then can be cloned into the amplification of nucleic acid produced by PCR using any well known method in this area can
In the cloning vector of duplication.
Once it is determined that the nucleotide sequence of antibody and corresponding amino acid sequence, it is possible to using well known in the art
The method of processing nucleotide sequence handles the nucleotide sequence of antibody to produce such as recombinant DNA technology, site-directed mutagenesis, PCR
Antibody with different aminoacids sequence, for example, produce 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, missing, and/or insert.
It can identify CDR's with the amino acid sequence of well known method inspection heavy chain and/or light variable domains
Sequence, methods described for example is compared to determine sequence by the known amino acid sequence with other heavy chains and light chain variable district
High variability region.Using conventional recombinant DNA technology, one or more CDR can be inserted into framework region, for example, be inserted into
It is as described above with humanizing non-human antibodies in people's framework region.Framework region can be framework region naturally occurring or shared, with
And preferably people's framework region is (see for example, 293Tthiaeta l., J.Mol.Biol.278:People in 457-479 (1998)
The list of framework region).Preferably, the polynucleotide encoding produced by framework region and CDR combination and the polypeptide of the present invention are passed through
The antibody of specific bond.Preferably, as discussed above, one or more 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors can be carried out in framework region,
And preferably described 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor improves antibody and the combination of its antigen.Furthermore it is possible to these methods to participating in chain
One or more variable region cysteine residues of interior disulfide bond are replaced or deleted, and are lacked with producing in one or more chains
The antibody molecule of disulfide bond.The present invention includes other changes to polynucleotides, this also those skilled in the art technical scope it
It is interior.
Present invention also offers a kind of recombinant expression carrier containing foregoing polynucleotides.
The recombination expression containing the nucleotide sequence for encoding binding molecule of the present invention can be prepared using well known technology to carry
Body.The expression vector includes the nucleotide sequence being operably connected with suitable transcription or translational regulation nucleotide sequence,
The transcription or translational regulation nucleotide sequence such as those sequences originating from mammal, microorganism, virus or insect genes
Row.The example of regulatory sequence includes transcripting promoter, operator, enhancer, mRNA ribosome bind sites, and/or other controls
Transcription and translation starting processed and the proper sequence terminated.When the regulatory sequence and the nucleotide sequence feature of suitable polypeptide
When related, nucleotide sequence is " operably connecting ".Therefore, if a promoter nucleotide sequence controls suitable core
The transcription of nucleotide sequence, then the promoter nucleotide sequence is exactly operably to be connected to such as antibody heavy chain sequence.
Present invention also offers a kind of place containing foregoing polynucleotides or foregoing recombinant expression carrier
Chief cell.
Host cell available for the present invention includes but is not limited to microorganism, such as through the restructuring containing antibody coding sequence
Bacterium (such as Escherichia coli (E.coli), the bacillus subtilis of phage DNA, DNA or cosmid DNA expression vectors conversion
Bacterium (B.subtilis))；Saccharomycete such as saccharomyces through the recombinant yeast expression vector conversion containing antibody coding sequence
(Saccharomyces), pichia (Pichia))；Through the recombinant virus expression vector containing antibody coding sequence (for example
Baculoviral) infection insect cell system；Through recombinant virus expression vector (such as cauliflower mosaic virus (CaMV)；Tobacco
Plant that is mosaic virus (TMV) infection or being converted through the recombinant plasmid expression vector (such as Ti-plasmids) containing antibody coding sequence
Thing cell system；Or carry containing from mammalian cell gene group promoter (such as metallothionein promoter) or
From the restructuring table of the promoter (for example, adenovirus late promoter, vaccinia virus 7.5K promoters) of mammalian virus
The mammalian cell system (such as COS, CHO, BHK, 293,3T3 cells) of expression constructs.
In specific embodiments of the present invention, the host cell is mammalian cell, more preferably CHO cells.
Converted with recombinant DNA, transfection host cell can be carried out with routine techniques well known to those skilled in the art.Some are adopted
Conversion, transfection method include but is not limited to：Conventional chemical processes such as calcium phosphate precipitation, PEI infection protocols, it is conventional
Mechanical means such as microinjection, electroporation, liposome packaging etc..
The transformant of acquisition can use conventional method culture, to express the binding molecule of the present invention.According to host used
Cell, culture medium used may be selected from various conventional mediums in culture.Trained under conditions of suitable for host cell growth
Support.After host cell growth is to appropriate cell density, is induced and selected with suitable method (such as temperature transition or chemical induction)
The promoter selected, a period of time is further cultured for by cell.
The binding molecule of the present invention is preferably produced using mammalian cell, and mammalian cell is usually required
Cultivated in culture medium containing serum.Need after the adaptation process to cell progress serum-free, cell can be allowed in serum-free
Normally grown in culture medium.
Suppress HCV the invention provides one kind, or, treat or prevent infection with hepatitis C virus disease
Pharmaceutical composition, described pharmaceutical composition includes the binding molecule of the invention of effective dose.
Present invention provides one or more components including one or more pharmaceutical compositions equipped with the present invention
The drug packages or kit of container.Optionally adjoint with these containers can be to manage the life of medicine or biological product
Production, using or sale government organs defineds form explanation, these explanations reflect the medicine that management human body is applied
Production, using or sale mechanism certification.
Product is detected present invention also offers a kind of HCV including binding molecule noted earlier.
The detection product includes but is not limited to detection reagent, kit, chip or test paper.It is every including knot noted earlier
The detection product for being capable of detecting when HCV for closing molecule is included within the scope of the present invention.
Present invention also offers a kind of method of the detection HCV levels of non-diagnostic purpose, it is characterised in that methods described bag
Include following steps：
(1) sample containing HCV is obtained；
(2) sample for obtaining step (1) is contacted with foregoing binding molecule；
(3) association reaction of detection sample and binding molecule.
Present invention also offers the method for the binding molecule described in preparation, it, which is included in, is suitable for causing from the coding present invention
Antibody molecule DNA marking proteins under conditions of culture comprising the present invention carrier host cell, and separation combine
Molecule.
Specifically, the invention provides a kind of preparation method of foregoing binding molecule, the preparation method is used
Be single B cell antibody technology of preparing.The single B cell antibody technology of preparing is to combine unicellular separation identification technology
A kind of vitro expression systems of monoclonal antibody of a variety of round pcr formation, i.e., directly obtain full people source from people's B cells
The method of monoclonal antibody.
Further, the preparation method comprises the following steps：
(1) HCV infection person's blood sample is gathered；
(2) separating peripheral blood mononuclear cells (PBMC)；
(3) PBMC cell sortings are carried out using flow cytometer, cell fragment are removed first, cell and dead cell is adhered,
CD3-/CD14-/CD16-/IgM- cell is obtained by fluorescent antibody staining, selection expression CD235a-/IgD-/CD20+'s
B cell, irises out CD27ALL memory B cells, and the target cell of E2 double fluorescence labelings is obtained with the antigen of specific marker fluorescein.
(4) antibody light chain and weight chain variable district in the single B cell of single-cell RT-PCR amplification step (2) acquisition are utilized
Nucleotide fragments；
(5) antibody light chain and the nucleotide fragments of weight chain variable district obtained step (3) is fused to containing human antibodies
Recombinant expression carrier is formed in the expression vector of constant region, host cell expression is imported afterwards；
(6) screening obtains the binding molecule of the invention with binding activity and neutralization activity.
Present invention also offers application of the foregoing binding molecule in HCV detection products are prepared.
The detection product includes foregoing binding molecule；It is described detection product include but is not limited to detection reagent,
Kit, chip or test paper.It is every to be capable of detecting when that HCV detection product is included in this including foregoing binding molecule
Within the scope of invention.
Present invention also offers application of the foregoing binding molecule in foregoing pharmaceutical composition is prepared.
Suppression HCV, or, prevention or treatment are being prepared present invention also offers foregoing binding molecule
Application in the pharmaceutical preparation of infection with hepatitis C virus disease.
Further, the disease includes hepatopathy, such as hepatitis, hepatic sclerosis or liver cancer；The exception of Extrahepatic diseases, such as skin, such as
Cryoglobulinemia, porphyria cutanea tarda, leukocytoclastic angiitis, livedo reticularis etc..
Further, the hepatitis is acute or chronic hepatitis C.
The binding molecule of the present invention can also have identical or complementary function Drug combination with other, and joint should
Effect can be binding molecule and other drugs function sum, can also be far longer than binding molecule and other drugs function
Sum, such case shows to generate synergy between the binding molecule and other drugs.
Term " monoclonal antibody " used herein refers to the antibody obtained from the substantially uniform colony of a class, except a small number of possible
Outside the mutation of the natural generation existed, the single antibody included in the colony is identical.Modifier " monoclonal " only represents anti-
The characteristic of body, is obtained from substantially uniform antibody population, and this can not be construed to need to produce anti-with any specific process
Body.
" variable " some parts for representing variable region in antibody of term used herein are different in sequence, and it is formed
Combinations and specificity of the various specific antibodies to its specific antigen.Changeability, which is concentrated in light chain and weight chain variable district, to be referred to as mutually
Mend in three fragments in decision area (CDR) or hypervariable region.Each self-contained four FR areas in the variable region of native heavy and light chain
(more conservative part in variable region), they are in generally beta sheet configuration, are connected by three CDR for forming connection ring, can shape
Into part β-pleated sheet structure.CDR in every chain is by FR areas firmly against together form together and with the CDR of another chain
The antigen-binding site of antibody.Constant region does not participate in the combination of antibody and antigen directly, but they show different effects
Function, such as participates in the cytotoxicity dependent on antibody of antibody.
As used herein, " effective dose " be mitigate enough one or more generally the symptom related to HCV infection or it is any by
HBV infection cause or associated disease or illness dosage.When considering preventative purposes, this term means pre- enough
Dosage that is anti-or delaying HCV infection to establish.
" sample " as described herein covers several samples type, includes the blood and other body fluid samples of biological origin
Product, solid tissue sample such as tissue biopsy sample either tissue culture or derived from cell therein or its offspring.Should
Term is additionally included in obtain after the sample that has been handled by any mode, such as with agent treatment, dissolve or be enriched with some
Composition such as protein or polynucleotides.The term covers the various clinical samples derived from any species, also including culture
Cell, cell conditioned medium and cell lysates.
As used herein, term " separation " refers to the protein isolated from its natural surroundings (that is, from least one
Separated in its natural adjoint other component).
All patents and publication being mentioned above are incorporated herein by reference in allowed by law degree, for
Describe and disclose the protein, enzyme, carrier, host cell and the side that are possibly used for the present invention reported in these patents and publication
The science of law.But present disclosure is understood not to recognize that the present invention is made the present invention can not take the lead in this and draped over one's shoulders due to formerly invention
Dew.
Brief description of the drawings
Fig. 1 is SDS-PAGE and Western-blot the detection figure of the TRN1008 antibody of the embodiment of the present invention, wherein A：
SDS-PAGE；B：Western-blot；
Fig. 2 schemes for the binding activity detection of the TRN1008 antibody and 8 kinds of different genotype HCV antigens of the embodiment of the present invention；
Fig. 3 is the TRN1008 antibody and 7 kinds of different subtype HCV euvirus neutralization activity design sketch of the embodiment of the present invention；
Fig. 4 for the embodiment of the present invention TRN1008 antibody and 4 kinds of different genotypes HCV antigen E2-core albumen parent
With determination of activity figure, wherein, A：E2-core-2a；B：E2-core-4a；C：E2-core-6a；D：E2-core-7a.
Specific embodiment
The invention is not restricted to particular methodology as described herein, scheme, cell line, carrier or reagent, because these are can
With change.In addition, terms used herein is intended merely to describe specific embodiment and the meaning of unrestricted the scope of the present invention.
Unless otherwise defined, then all technologies used herein and academic term and any abbreviation are respectively provided with and this area skill
The implication identical implication that art personnel are commonly understood by.Although implement the present invention when can use with it is described herein similar or identical
Any method and material, but also depict using equipment and material herein.
The monoclonal neutralizing antibody TRN1008 of the full people source HCV-Ab IgG of embodiment 1 preparation
1st, sample acquisition：HCV infection person's anticoagulation cirumferential blood picks up from Yunnan Province Kunming, and the patient confirms that infection time is
2010.
2nd, memory B cells are sorted
Gather HCV infection person's blood sample, separating peripheral blood mononuclear cells (Peripheral blood
Mononuclear cell, PBMC), BD FACSria flow cytometers (BD Biosciences, San Jose, CA) are carried out
Cell sorting, first remove cell fragment, be adhered cell and dead cell, by fluorescent antibody staining obtain CD3-/CD14-/
CD16-/IgM- cell, selection expression CD235a-/IgD-/CD20+ B cell, irises out CD27ALL memory B cells, with spy
The antigen of different in nature mark fluorescent element obtains the target cell of E2 double fluorescence labelings.
3rd, single-cell RT-PCR
0.5 μM of each subtype heavy chain and light chain will be added in the mixed liquid of PCR predictions of 96 orifice plates containing single bone-marrow-derived lymphocyte
Constant region primers and Superscript IV reverse transcriptases (Invitrogen, Carlsbad, CA), while setting positive and cloudy
Property control；Reverse transcription PCR condition：55 DEG C of 60min, are down to 4 DEG C.Product is cDNA-20 DEG C long-term to be preserved.
4th, the amplification of antibody variable region target gene
With reverse transcription product (cDNA) for template, addition AmpliTaq Gold 360Master Mix (Invitrogen,
Carlsbad, CA), and 0.5 μM of each subtype heavy chain and the specific primer of light chain antibody.Reaction condition：95 DEG C of pre-degeneration
5min, then carries out 35 PCR cycles, and each circulation is：94 DEG C × 30s, 58/60/64 (H/K/L) DEG C × 30s, 72 DEG C ×
1.5min, 10 DEG C are down to after extending 7min with 72 DEG C.
5th, DNA gel electroresis appraisal
Electrophoresis experiment is carried out using 2% Ago-Gel to be identified.Take 2 μ L PCR reaction products and 18 μ L's 0.1%
Loading after Loading Buffer are mixed, carries out electrophoresis 12min under the EG patterns of E-base electrophoresis systems.Gel imaging system
System detection PCR primer size.
6th, the structure of the expression vector of recombinant antibodies
With specific primer obtain ELISA test positive heavy chain of antibody and light chain gene segment (including variable region and
Constant region), heavy chain and light chain gene are linked on pcDNA3.3 carriers by the method cloned using TA respectively, by connection product
Convert in DH5 α competence bacteriums, 37 DEG C of overnight incubations on the flat board containing ampicillin, immediately 12 single bacterium colonies of picking
Enter performing PCR identification (PCR reaction conditions with specific primer：94 DEG C of pre-degenerations 3min, 94 DEG C of denaturation 30s, 55 DEG C of annealing 30s,
72 DEG C of extension 1min40s, 28 circulations, last 72 DEG C re-extend 5min), take 2 μ LPCR products enterprising in 1% Ago-Gel
Row electrophoresis detection, chooses the bacterium colony progress gene sequencing that PCR is accredited as the positive, comparison result is correctly heavy chain of antibody and light
The recombinant expression plasmid of chain.
7th, the expression of ELISA testing goals gene
The plasmid of a large amount of amplification expression positive antibody heavy chains and light chain gene, endotoxin-free extracting in bacillus coli DH 5 alpha
Kit is extracted.With transfection reagent Polyetherimide corotation transfected cho cells, 4-6 hours addition fresh serum frees after transfection
Culture medium, is placed in 37 DEG C, 8%CO2Cultivated 96 hours in constant incubator, collect cell conditioned medium and detected.
Cultivate the antibody that cell conditioned medium after 72h uses direct competive ELISA testing goal gene expression.ELISA is screened：
Using different HCV envelope glycoproteins as antigen, and it is 100ng/ml by antigen diluent concentration with coating buffer, is coated in ELISA 96
Orifice plate, per the μ l of hole 100,4 DEG C overnight.37 DEG C of 2 hours of closing of confining liquid.Add primary antibody after closing, antibody initial concentration is 25 μ g/
Ml, 3 times of gradient dilutions are 100 μ l, 37 DEG C of 1 hours of incubation, while being used as the positive with HCV positive patients serum per pore volume
Control, Rabies virus antibody is negative control.The goat anti-human igg (1 marked with HRP:2000 dilutions) it is 1 small as 37 DEG C of incubations of secondary antibody
When.Add after substrate nitrite ion (TMB), 37 DEG C of avoid light place 5min, with 2M sulfuric acid stopped reactions, compared with 450nm wavelength
Color.
8th, in antibody and test
The ELISA antibody for having binding activity screened is subjected to neutralization experiment.By the volume of different HCV envelope glycoproteins
Code sequence is packaged into plasmid, and adding fluorescein Luciferase genes transfection 293T cells is used to pack HCV virus (HCVpp),
Collecting supernatant is used to infect.Huh7 cells are spread in 96 orifice plates, per hole cell about 1*104It is individual, it is 100 μ l per pore volume, overnight
Culture, cell about 30% is paved with during infection.HCVpp is mixed with antibody, and room temperature places 30min.HCVpp is added to mix with antibody
Liquid infects Huh7 cells in 96 orifice plates.Control group changes liquid after only adding HCVpp, infection 24h, continues to cultivate 1-2 days.After infection
Luciferase activity is determined after 2-3 days, measuring method is removes cell conditioned medium, per hole with 30 μ l lysates, fully after cracking
20 μ l cell pyrolysis liquids plus 30 μ l substrates are taken, fluorescent value is read.Compare antibody and control group, calculate and neutralize efficiency.
9th, antibody great expression and purifying
The expression vector of having of going out of the experimental identification heavy chain of antibody that the numbering of neutralization activity is TRN1008 and light chain will be neutralized
(wherein, the amino acid sequence of weight chain variable district such as SEQ ID NO:Shown in 1；The amino acid sequence of antibody light chain variable region such as SEQ
ID NO:Shown in 5) (cell density reaches 1.6 × 10 to corotation transfected cho cell6Individual/ml), mend culture medium within 6-8 hours after transfection,
37 DEG C, 8%CO2, cultivate 120 hours in 125rpm shaking tables.Then transfection supernatant is collected, 4000rp, is centrifuged 1 hour, abandoned by 4 DEG C
Precipitation, is purified using albumen (Protein) A affinity chromatographies.Antibody is examined by SDS-PAGE and Western-blot
TRN1008 expression and purification result, as a result as shown in figure 1, obtaining compared with pure protein, and can be clearly observable the antibody after unwinding
Light and heavy chain.
The binding activity detection of the TRN1008 antibody of embodiment 2
1st, the specific binding detection of TRN1008 antibody and HCV envelope glycoproteins
The ELISA method mentioned with embodiment 1 is detected to the binding specificity of the antibody of expression and purification：Will be different
HCV envelope glycoprotein coded sequences are packaged into plasmid, and adding fluorescein Luciferase genes transfection 293T cells is used to pack
HCV virus (HCVpp), collecting supernatant is used to infect.Huh7 cells are spread in 96 orifice plates, per hole cell about 1*104It is individual, per hole body
It is 100 μ l to accumulate, incubated overnight, and cell about 30% is paved with during infection.HCVpp is mixed with the antibody HCV of various concentrations, initial concentration
75 μ g/ml, 3 times of dilutions, room temperature places 30min.HCVpp is added with antibody HCV mixed liquors in 96 orifice plates, Huh7 cells are infected.
Control group changes liquid after only adding HCVpp, infection 24h, continues to cultivate 1-2 days.Luciferase is determined after infection after 2-3 days to live
Property, measuring method is removes cell conditioned medium, per hole with 30 μ l lysates, fully takes 20 μ l cell pyrolysis liquids plus 30 μ l bottoms after cracking
Thing, reads fluorescent value.
As a result：TRN1008 antibody energy 100% is specifically bound with HCV envelope glycoproteins, fluorescent value reduction.
2nd, the binding activity detection of TRN1008 antibody and different genotype HCV antigens
Using 8 kinds of different genotype HCV envelope glycoproteins as antigen, and it is 100ng/ by antigen diluent concentration with coating buffer
Ml, is coated in the orifice plates of ELISA 96, and per the μ l of hole 100,4 DEG C overnight.37 DEG C of 2 hours of closing of confining liquid.Add primary antibody after closing, often
Pore volume is 100 μ l, 37 DEG C of 1 hours of incubation.The goat anti-human igg (1 marked with HRP:2000 dilution) as secondary antibody 37 DEG C incubate
Educate 1 hour.Add after substrate nitrite ion (TMB) 100 μ L/ holes, 37 DEG C of avoid light place 5min, with 2M sulfuric acid stopped reactions, use
450nm wavelength carries out colorimetric.
As a result as shown in Fig. 2 TRN1008 antibody can be with different genotype HCV antigen bindings, and TRN1008 knot
Close activity extremely strong, 1b, 1a in wherein Fig. 2,2,3,4,5,6,7 represent respectively HCV1b, HCV1a, HCV2, HCV3, HCV4,
HCV5、HCV6、HCV7。
The TRN1008 antibody of embodiment 3 is detected from the neutralization activity of different HCV euviruses strains
Huh7 cells are spread in 96 orifice plates, incubated overnight, cell about 30% is paved with during infection.The strain of HCV euviruses (h77,
Con1, JFH1, S52, ED43, SA13, HK6a) mixed with the antibody of various concentrations, the μ g/ml of initial concentration 25,3 times of dilutions, room
Temperature places 30min.HCV virus strain (h77, Con1, JFH1, S52, ED43, SA13, HK6a) is added with antibody mixed liquor in 96
Orifice plate, infects Huh7 cells.Control group changes liquid after only adding HCV virus strain 2G9, infection 24h, continues to cultivate 1-2 days.After infection
Activity is determined after 2-3 days, fluorescent value is read.Compare antibody and control group, calculate and neutralize efficiency.
As a result as shown in figure 3, TRN1008 antibody see the table below from the neutralization efficiency of different HCV virus strains, TRN1008 resists
Body is in HCV euviruses strain H77 (1a), Con1 (1b), JFH1 (2a), S52 (3a), ED43 (4a), SA13 (5a) and HK6a
It is all very good with efficiency, its applications well prospect in immunization therapy and prevention can be shown.
The neutralization efficiency of the TRN1008 antibody of table 1 and different HCV virus strains
The affine Activity determination of the TRN1008 antibody of embodiment 4 and HCV envelope glycoprotein
The parent that TRN1008 and HCV envelope glycoprotein (HCV-E2-core, antigen) is combined is carried out with BiaCore X-100
With power and dynamic analysis：HCV-E2-core albumen is diluted as analyte with HBS-EP buffer solutions, and analyte is gradually to increase
Concentration flow successively through chip, respectively obtain signal curve.BiaCore X-100System softwares are analyzed, and then are obtained
TRN1008 affinity and kinetic parameter result.
As a result as shown in Fig. 4 and table 2.TRN1008 antibody and antigen E2-core-2a, E2-core-4a, E2-core-6a
Be respectively 1.32E-11M, 1.12E-9M, 2E-11M, 2.98E-12M with E2-core-7a KD values, display TRN1008 have compared with
High affinity activity.
The affinity kinetic results parameter list of the TRN1008 antibody of table 2 and HCV virus antigen
| Antigen | ka(1/Ms) | kd(1/s) | KD(M) |
| E2-core-2a | 13010 | 1.72E-07 | 1.32E-11 |
| E2-core-4a | 3.48E+04 | 3.89E-05 | 1.12E-09 |
| E2-core-6a | 24410 | 4.87E-07 | 2E-11 |
| E2-core-7a | 4.06E+04 | 1.21E-07 | 2.98E-12 |
Although above only describes the embodiment example of the present invention, those skilled in the art should manage
Solution, these are merely illustrative of, and protection scope of the present invention is defined by the appended claims.Those skilled in the art
On the premise of the principle and essence without departing substantially from the present invention, various changes or modifications, but this can be made to these embodiments
A little changes or modification each fall within protection scope of the present invention.
SEQUENCE LISTING
<110>Guangzhou Tylenol enlightening bio tech ltd
Zhuhai Tylenol Mai Bo Bioisystech Co., Ltd
Ji'nan University
<120>A kind of preparation method and purposes for treating hepatitis C virus cytotoxic drug
<160> 8
<170> PatentIn version 3.5
<210> 1
<211> 123
<212> PRT
<213>People source
<400> 1
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Pro Ser Gly Gly Ser Phe Thr Gly His
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Pro Glu Trp Met
35 40 45
Gly Cys Ile Asn Pro Asn Ser Gly Glu Thr Asn Tyr Glu Gln Lys Phe
50 55 60
Arg Gly Arg Val Thr Leu Thr Arg Asp Thr Ser Ile Asn Thr Ala Tyr
65 70 75 80
Met Glu Val Ser Ser Leu Thr Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Thr Pro Gly Glu Gly Thr Thr Pro Phe Tyr Phe Gly Met Asp Val
100 105 110
Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
115 120
<210> 2
<211> 8
<212> PRT
<213>People source
<400> 2
Gly Gly Ser Phe Thr Gly His Tyr
1 5
<210> 3
<211> 8
<212> PRT
<213>People source
<400> 3
Ile Asn Pro Asn Ser Gly Glu Thr
1 5
<210> 4
<211> 16
<212> PRT
<213>People source
<400> 4
Ala Thr Pro Gly Glu Gly Thr Thr Pro Phe Tyr Phe Gly Met Asp Val
1 5 10 15
<210> 5
<211> 107
<212> PRT
<213>People source
<400> 5
Asp Ile Val Met Thr Gln Ser Pro Pro Ser Val Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Thr Gln Gly Ala Ser Thr Trp
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Glu Leu Leu Ile
35 40 45
Tyr Ala Thr Asn Ile Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Thr Asn Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Ile Lys Arg Phe Pro Leu
85 90 95
Thr Phe Gly Gly Gly Thr Lys Val Asp Ile Lys
100 105
<210> 6
<211> 6
<212> PRT
<213>People source
<400> 6
Gln Gly Ala Ser Thr Trp
1 5
<210> 7
<211> 3
<212> PRT
<213>People source
<400> 7
Ala Thr Asn
1
<210> 8
<211> 9
<212> PRT
<213>People source
<400> 8
Gln His Ile Lys Arg Phe Pro Leu Thr
1 5
Claims (10)
1. a kind of binding molecule of separation, it is characterised in that the binding molecule includes：
(1)SEQ ID NO:Heavy chain CDR1, SEQ ID NO shown in 2:Heavy chain CDR2, SEQ ID NO shown in 3:Shown in 4
Heavy chain CDR3；And/or
(2)SEQ ID NO:Light chain CDR1, SEQ ID NO shown in 6:Light chain CDR2, SEQ ID NO shown in 7:Shown in 8
Light chain CDR3.
2. binding molecule according to claim 1, it is characterised in that the binding molecule includes：
(1) weight chain variable district, the weight chain variable district has SEQ ID NO:Amino acid sequence shown in 1；And/or
(2) light chain variable district, the light chain variable district has SEQ ID NO:Amino acid sequence shown in 5.
3. binding molecule according to claim 1, it is characterised in that the binding molecule is antibody molecule or its immunology
Active fragment.
4. encode the polynucleotides of the binding molecule any one of claim 1-3.
5. a kind of recombinant expression carrier of the polynucleotides including described in claim 4.
6. a kind of host cell, it is characterised in that the polynucleotides or right that the host cell contains described in claim 4 will
Seek the recombinant expression carrier described in 5.
7. the pharmaceutical composition or HCV of the binding molecule any one of a kind of claim 1-3 including therapeutically effective amount
Detect product.
8. the method for the detection HCV levels of a kind of non-diagnostic purpose, it is characterised in that methods described comprises the following steps：
(1) sample containing HCV is obtained；
(2) sample for obtaining step (1) is contacted with the binding molecule any one of claim 1-3；
(3) detection sample and claim 1-3 any one of binding molecule combination situation.
9. the binding molecule any one of claim 1-3 is preparing HCV detection products or is suppressing HCV medicine or pre-
Application in anti-or treatment disease as caused by HCV infection medicine.
10. application according to claim 9, it is characterised in that the disease as caused by HCV infection includes hepatopathy, such as
Hepatitis, hepatic sclerosis or liver cancer；It is the exception of Extrahepatic diseases, such as skin, such as cryoglobulinemia, porphyria cutanea tarda, white thin
Born of the same parents' brokenness vasculitis, livedo reticularis.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710668064.2A CN107216388B (en) | 2017-08-07 | 2017-08-07 | Preparation method and application of medicine for treating hepatitis C virus |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710668064.2A CN107216388B (en) | 2017-08-07 | 2017-08-07 | Preparation method and application of medicine for treating hepatitis C virus |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107216388A true CN107216388A (en) | 2017-09-29 |
| CN107216388B CN107216388B (en) | 2020-05-05 |
Family
ID=59954966
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710668064.2A Active CN107216388B (en) | 2017-08-07 | 2017-08-07 | Preparation method and application of medicine for treating hepatitis C virus |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107216388B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7605237B2 (en) * | 2006-10-02 | 2009-10-20 | Regeneron Pharmaceuticals, Inc. | High affinity human antibodies to human IL-4 receptor |
| CN106749644A (en) * | 2016-11-14 | 2017-05-31 | 广州泰诺迪生物科技有限公司 | A kind of neutralizing antibody TRN1001 of full people source HCV-Ab IgG |
-
2017
- 2017-08-07 CN CN201710668064.2A patent/CN107216388B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7605237B2 (en) * | 2006-10-02 | 2009-10-20 | Regeneron Pharmaceuticals, Inc. | High affinity human antibodies to human IL-4 receptor |
| CN106749644A (en) * | 2016-11-14 | 2017-05-31 | 广州泰诺迪生物科技有限公司 | A kind of neutralizing antibody TRN1001 of full people source HCV-Ab IgG |
Non-Patent Citations (1)
| Title |
|---|
| GENBANK: "Sequence 10453 from US9012723;GenBank：AKY17227.1", 《NCBI》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107216388B (en) | 2020-05-05 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102272155B (en) | Dengue fever virus neutralizing antibody and application thereof | |
| CN113336844B (en) | Shark single domain antibody targeting novel coronavirus N protein, and preparation method and application thereof | |
| CN113264998A (en) | Single-chain antibody of S1 protein on surface of anti-new coronavirus SARS-CoV-2 and application thereof | |
| CN107226861A (en) | The anti-H7N9 avian influenza virus neutrality antibody 1F7L in people source and its application | |
| CN104603149B (en) | The composition related to prevention and treatment rabies infection and method | |
| CN107043425B (en) | anti-PD 1 and CD20 bispecific antibodies and uses thereof | |
| CN109776678A (en) | A kind of humanization PD-L1 monoclonal antibody, preparation method and application | |
| CN110317267A (en) | For the bispecific antibody and application thereof of hydrophobin | |
| JP2005531286A (en) | Human monoclonal antibody Fab fragment directed against HCVE2 glycoprotein and having in vitro neutralizing activity | |
| CN106749644B (en) | A kind of neutralizing antibody-TRN1001 of full people source HCV-Ab IgG | |
| CN110494163A (en) | Blood coagulation factor VIII simulated albumin and application thereof | |
| CN114573691B (en) | Humanized neutralizing antibody or antigen binding fragment thereof and application thereof | |
| MX2011006722A (en) | Anti-hcv monoclonal antibody as a medicament for the therapeutic treatment and prevention of hcv infections. | |
| CN107383199A (en) | A kind of monoclonal antibody of S adenomethionine synthases and its application | |
| CN105061590B (en) | For the bispecific antibody and application thereof of hepatitis B surface albumen | |
| CN106749645B (en) | A kind of neutralizing antibody of full source of people anti-hepatitis c virus | |
| CN107216388A (en) | A kind of preparation method and purposes for treating hepatitis C virus cytotoxic drug | |
| CN109705219A (en) | Monoclonal antibody in conjunction with mesothelin and preparation method thereof | |
| CN106831987A (en) | A kind of anti-human HER2 antibody and its encoding gene and application | |
| CN112159470B (en) | anti-WNV (WNV) infection binding molecule | |
| CN101538328B (en) | Humanized neutralizing engineered antibody for targeting avian influenza virus H5N1 | |
| CN108929382B (en) | The antibody of anti-SERINC5 a kind of and its application | |
| US10888615B2 (en) | Neutralizing human monoclonal antibody 8D6 against HCV infection | |
| CN101591396B (en) | Anti-erbB2 human antibody MIL-5 and application thereof | |
| CN110272497A (en) | A kind of anti-cyclic citrullinated peptide tetravalence small molecular antibody of high-affinity and the preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CP01 | Change in the name or title of a patent holder |
Address after: 510300 unit 207, second floor, office area, No. 1, helix 4th Road, International Biological Island, Guangzhou, Guangdong Patentee after: GUANGZHOU TAINUODI BIOTECHNOLOGY Co.,Ltd. Patentee after: Zhuhai Tainuo Maibo Pharmaceutical Co.,Ltd. Patentee after: Jinan University Address before: 510300 unit 207, second floor, office area, No. 1, helix 4th Road, International Biological Island, Guangzhou, Guangdong Patentee before: GUANGZHOU TAINUODI BIOTECHNOLOGY Co.,Ltd. Patentee before: ZHUHAI TRINOMAB BIOTECHNOLOGY Co.,Ltd. Patentee before: Jinan University |
|
| CP01 | Change in the name or title of a patent holder | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20230818 Address after: 519090 room 110, first floor, plant 6, No. 6366, Zhuhai Avenue, Jinwan District, Zhuhai City, Guangdong Province Patentee after: Zhuhai Tainuo Maibo Pharmaceutical Co.,Ltd. Patentee after: Jinan University Address before: 510300 unit 207, second floor, office area, No. 1, helix 4th Road, International Biological Island, Guangzhou, Guangdong Patentee before: GUANGZHOU TAINUODI BIOTECHNOLOGY Co.,Ltd. Patentee before: Zhuhai Tainuo Maibo Pharmaceutical Co.,Ltd. Patentee before: Jinan University |
|
| TR01 | Transfer of patent right |