CN109824777A - A kind of monoclonal antibody and its preparation method and application with inhibition vitreous-body-retina fibrosis lesion effect - Google Patents
A kind of monoclonal antibody and its preparation method and application with inhibition vitreous-body-retina fibrosis lesion effect Download PDFInfo
- Publication number
- CN109824777A CN109824777A CN201910197628.8A CN201910197628A CN109824777A CN 109824777 A CN109824777 A CN 109824777A CN 201910197628 A CN201910197628 A CN 201910197628A CN 109824777 A CN109824777 A CN 109824777A
- Authority
- CN
- China
- Prior art keywords
- antibody
- mouse
- monoclonal
- vitreous
- ctgf
- 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.)
- Pending
Links
- 206010016654 Fibrosis Diseases 0.000 title claims abstract description 31
- 230000004761 fibrosis Effects 0.000 title claims abstract description 31
- 230000003902 lesion Effects 0.000 title claims abstract description 25
- 238000002360 preparation method Methods 0.000 title claims abstract description 20
- 230000000694 effects Effects 0.000 title claims abstract description 19
- 230000005764 inhibitory process Effects 0.000 title description 6
- 101000777550 Homo sapiens CCN family member 2 Proteins 0.000 claims abstract description 89
- 102100031168 CCN family member 2 Human genes 0.000 claims abstract description 67
- 108010021625 Immunoglobulin Fragments Proteins 0.000 claims abstract description 7
- 102000008394 Immunoglobulin Fragments Human genes 0.000 claims abstract description 7
- 230000002401 inhibitory effect Effects 0.000 claims abstract description 7
- 210000004027 cell Anatomy 0.000 claims description 39
- 102000047612 human CCN2 Human genes 0.000 claims description 22
- 210000004408 hybridoma Anatomy 0.000 claims description 15
- 238000002649 immunization Methods 0.000 claims description 15
- 206010003445 Ascites Diseases 0.000 claims description 14
- 238000000034 method Methods 0.000 claims description 13
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 12
- 238000001514 detection method Methods 0.000 claims description 11
- 201000010099 disease Diseases 0.000 claims description 11
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 10
- 230000004927 fusion Effects 0.000 claims description 9
- 108090000623 proteins and genes Proteins 0.000 claims description 9
- 206010035226 Plasma cell myeloma Diseases 0.000 claims description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 8
- 239000007983 Tris buffer Substances 0.000 claims description 8
- 239000002671 adjuvant Substances 0.000 claims description 8
- 238000010828 elution Methods 0.000 claims description 8
- 201000000050 myeloid neoplasm Diseases 0.000 claims description 8
- 102000004169 proteins and genes Human genes 0.000 claims description 8
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 7
- 238000002347 injection Methods 0.000 claims description 7
- 239000007924 injection Substances 0.000 claims description 7
- 238000011081 inoculation Methods 0.000 claims description 7
- 150000007523 nucleic acids Chemical group 0.000 claims description 7
- 238000000502 dialysis Methods 0.000 claims description 5
- 238000001914 filtration Methods 0.000 claims description 5
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 5
- 238000000926 separation method Methods 0.000 claims description 5
- 238000000338 in vitro Methods 0.000 claims description 4
- 208000014674 injury Diseases 0.000 claims description 4
- 239000011259 mixed solution Substances 0.000 claims description 4
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 4
- 239000011780 sodium chloride Substances 0.000 claims description 4
- 241001465754 Metazoa Species 0.000 claims description 3
- 125000000129 anionic group Chemical group 0.000 claims description 3
- 239000003795 chemical substances by application Substances 0.000 claims description 3
- 239000003814 drug Substances 0.000 claims description 3
- 229940079593 drug Drugs 0.000 claims description 3
- 239000003480 eluent Substances 0.000 claims description 3
- 230000004048 modification Effects 0.000 claims description 3
- 238000012986 modification Methods 0.000 claims description 3
- 230000008733 trauma Effects 0.000 claims description 3
- 238000012216 screening Methods 0.000 claims description 2
- 206010012689 Diabetic retinopathy Diseases 0.000 claims 1
- 230000003014 reinforcing effect Effects 0.000 claims 1
- 238000001356 surgical procedure Methods 0.000 claims 1
- 210000004369 blood Anatomy 0.000 abstract description 4
- 239000008280 blood Substances 0.000 abstract description 4
- 102000001706 Immunoglobulin Fab Fragments Human genes 0.000 abstract description 2
- 108010054477 Immunoglobulin Fab Fragments Proteins 0.000 abstract description 2
- 210000001124 body fluid Anatomy 0.000 abstract description 2
- 239000010839 body fluid Substances 0.000 abstract description 2
- 229940125645 monoclonal antibody drug Drugs 0.000 abstract 1
- 239000012071 phase Substances 0.000 description 13
- 238000001962 electrophoresis Methods 0.000 description 10
- 239000000047 product Substances 0.000 description 9
- 238000000746 purification Methods 0.000 description 9
- 230000000007 visual effect Effects 0.000 description 8
- 238000005119 centrifugation Methods 0.000 description 7
- 239000001963 growth medium Substances 0.000 description 7
- 239000007788 liquid Substances 0.000 description 7
- 235000018102 proteins Nutrition 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- 102000036639 antigens Human genes 0.000 description 6
- 108091007433 antigens Proteins 0.000 description 6
- 230000003053 immunization Effects 0.000 description 6
- 238000002965 ELISA Methods 0.000 description 5
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical class N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 5
- 239000000427 antigen Substances 0.000 description 5
- 230000003176 fibrotic effect Effects 0.000 description 5
- 210000001519 tissue Anatomy 0.000 description 5
- 102000015225 Connective Tissue Growth Factor Human genes 0.000 description 4
- 108010039419 Connective Tissue Growth Factor Proteins 0.000 description 4
- 239000002202 Polyethylene glycol Substances 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 239000008363 phosphate buffer Substances 0.000 description 4
- 229920001223 polyethylene glycol Polymers 0.000 description 4
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- 238000007920 subcutaneous administration Methods 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 108020004414 DNA Proteins 0.000 description 3
- 208000010412 Glaucoma Diseases 0.000 description 3
- 102000004887 Transforming Growth Factor beta Human genes 0.000 description 3
- 108090001012 Transforming Growth Factor beta Proteins 0.000 description 3
- 102000009618 Transforming Growth Factors Human genes 0.000 description 3
- 150000001413 amino acids Chemical class 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 230000006378 damage Effects 0.000 description 3
- 239000000835 fiber Substances 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 230000006785 proliferative vitreoretinopathy Effects 0.000 description 3
- 108700038606 rat Smooth muscle Proteins 0.000 description 3
- 238000005215 recombination Methods 0.000 description 3
- 230000006798 recombination Effects 0.000 description 3
- ZRKFYGHZFMAOKI-QMGMOQQFSA-N tgfbeta Chemical compound C([C@H](NC(=O)[C@H](C(C)C)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCSC)C(C)C)[C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O)C1=CC=C(O)C=C1 ZRKFYGHZFMAOKI-QMGMOQQFSA-N 0.000 description 3
- ULXYQAJWJGLCNR-YUMQZZPRSA-N Leu-Asp-Gly Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(O)=O ULXYQAJWJGLCNR-YUMQZZPRSA-N 0.000 description 2
- PNUCWVAGVNLUMW-CIUDSAMLSA-N Leu-Cys-Ser Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CS)C(=O)N[C@@H](CO)C(O)=O PNUCWVAGVNLUMW-CIUDSAMLSA-N 0.000 description 2
- BAVYZALUXZFZLV-UHFFFAOYSA-N Methylamine Chemical compound NC BAVYZALUXZFZLV-UHFFFAOYSA-N 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 208000002158 Proliferative Vitreoretinopathy Diseases 0.000 description 2
- 206010038934 Retinopathy proliferative Diseases 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 2
- IRLYZKKNBFPQBW-XGEHTFHBSA-N Val-Cys-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CS)NC(=O)[C@H](C(C)C)N)O IRLYZKKNBFPQBW-XGEHTFHBSA-N 0.000 description 2
- 208000027418 Wounds and injury Diseases 0.000 description 2
- 210000001015 abdomen Anatomy 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 230000004888 barrier function Effects 0.000 description 2
- 210000001185 bone marrow Anatomy 0.000 description 2
- 230000007910 cell fusion Effects 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000004438 eyesight Effects 0.000 description 2
- 206010020718 hyperplasia Diseases 0.000 description 2
- FDGQSTZJBFJUBT-UHFFFAOYSA-N hypoxanthine Chemical compound O=C1NC=NC2=C1NC=N2 FDGQSTZJBFJUBT-UHFFFAOYSA-N 0.000 description 2
- 238000003119 immunoblot Methods 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 238000007912 intraperitoneal administration Methods 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- 210000004698 lymphocyte Anatomy 0.000 description 2
- 108010038320 lysylphenylalanine Proteins 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 208000021971 neovascular inflammatory vitreoretinopathy Diseases 0.000 description 2
- 238000006386 neutralization reaction Methods 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 239000012474 protein marker Substances 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 210000001525 retina Anatomy 0.000 description 2
- 230000028327 secretion Effects 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 210000000329 smooth muscle myocyte Anatomy 0.000 description 2
- 210000004988 splenocyte Anatomy 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 229940104230 thymidine Drugs 0.000 description 2
- -1 trihydroxy methyl amino Chemical group 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- XEXJJJRVTFGWIC-FXQIFTODSA-N Ala-Asn-Arg Chemical compound C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)N XEXJJJRVTFGWIC-FXQIFTODSA-N 0.000 description 1
- OILNWMNBLIHXQK-ZLUOBGJFSA-N Ala-Cys-Ser Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CS)C(=O)N[C@@H](CO)C(O)=O OILNWMNBLIHXQK-ZLUOBGJFSA-N 0.000 description 1
- SDZRIBWEVVRDQI-CIUDSAMLSA-N Ala-Lys-Asp Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(O)=O SDZRIBWEVVRDQI-CIUDSAMLSA-N 0.000 description 1
- PIXQDIGKDNNOOV-GUBZILKMSA-N Ala-Lys-Gln Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(N)=O)C(O)=O PIXQDIGKDNNOOV-GUBZILKMSA-N 0.000 description 1
- MAZZQZWCCYJQGZ-GUBZILKMSA-N Ala-Pro-Arg Chemical compound [H]N[C@@H](C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCNC(N)=N)C(O)=O MAZZQZWCCYJQGZ-GUBZILKMSA-N 0.000 description 1
- AUFACLFHBAGZEN-ZLUOBGJFSA-N Ala-Ser-Cys Chemical compound N[C@@H](C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CS)C(=O)O AUFACLFHBAGZEN-ZLUOBGJFSA-N 0.000 description 1
- MTDDMSUUXNQMKK-BPNCWPANSA-N Ala-Tyr-Arg Chemical compound C[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)N MTDDMSUUXNQMKK-BPNCWPANSA-N 0.000 description 1
- VHAQSYHSDKERBS-XPUUQOCRSA-N Ala-Val-Gly Chemical compound C[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)NCC(O)=O VHAQSYHSDKERBS-XPUUQOCRSA-N 0.000 description 1
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 1
- DFCIPNHFKOQAME-FXQIFTODSA-N Arg-Ala-Asn Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(N)=O)C(O)=O DFCIPNHFKOQAME-FXQIFTODSA-N 0.000 description 1
- VBFJESQBIWCWRL-DCAQKATOSA-N Arg-Ala-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CCCNC(N)=N VBFJESQBIWCWRL-DCAQKATOSA-N 0.000 description 1
- YWENWUYXQUWRHQ-LPEHRKFASA-N Arg-Cys-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CS)NC(=O)[C@H](CCCN=C(N)N)N)C(=O)O YWENWUYXQUWRHQ-LPEHRKFASA-N 0.000 description 1
- NIUDXSFNLBIWOB-DCAQKATOSA-N Arg-Leu-Cys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CCCN=C(N)N)N NIUDXSFNLBIWOB-DCAQKATOSA-N 0.000 description 1
- OTZMRMHZCMZOJZ-SRVKXCTJSA-N Arg-Leu-Glu Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O OTZMRMHZCMZOJZ-SRVKXCTJSA-N 0.000 description 1
- NMRHDSAOIURTNT-RWMBFGLXSA-N Arg-Leu-Pro Chemical compound CC(C)C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CCCN=C(N)N)N NMRHDSAOIURTNT-RWMBFGLXSA-N 0.000 description 1
- BSYKSCBTTQKOJG-GUBZILKMSA-N Arg-Pro-Ala Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C)C(O)=O BSYKSCBTTQKOJG-GUBZILKMSA-N 0.000 description 1
- FTMRPIVPSDVGCC-GUBZILKMSA-N Arg-Val-Cys Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CCCN=C(N)N)N FTMRPIVPSDVGCC-GUBZILKMSA-N 0.000 description 1
- QLSRIZIDQXDQHK-RCWTZXSCSA-N Arg-Val-Thr Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O QLSRIZIDQXDQHK-RCWTZXSCSA-N 0.000 description 1
- PIWWUBYJNONVTJ-ZLUOBGJFSA-N Asn-Asp-Asn Chemical compound C([C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CC(=O)N)C(=O)O)N)C(=O)N PIWWUBYJNONVTJ-ZLUOBGJFSA-N 0.000 description 1
- NKTLGLBAGUJEGA-BIIVOSGPSA-N Asn-Cys-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CS)NC(=O)[C@H](CC(=O)N)N)C(=O)O NKTLGLBAGUJEGA-BIIVOSGPSA-N 0.000 description 1
- SPIPSJXLZVTXJL-ZLUOBGJFSA-N Asn-Cys-Ser Chemical compound NC(=O)C[C@H](N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CO)C(O)=O SPIPSJXLZVTXJL-ZLUOBGJFSA-N 0.000 description 1
- NNDSLVWAQAUPPP-GUBZILKMSA-N Asn-Met-Met Chemical compound CSCC[C@@H](C(=O)N[C@@H](CCSC)C(=O)O)NC(=O)[C@H](CC(=O)N)N NNDSLVWAQAUPPP-GUBZILKMSA-N 0.000 description 1
- ZEDBMCPXPIYJLW-XHNCKOQMSA-N Asp-Glu-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CC(=O)O)N)C(=O)O ZEDBMCPXPIYJLW-XHNCKOQMSA-N 0.000 description 1
- YNCHFVRXEQFPBY-BQBZGAKWSA-N Asp-Gly-Arg Chemical compound OC(=O)C[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCCN=C(N)N YNCHFVRXEQFPBY-BQBZGAKWSA-N 0.000 description 1
- PYXXJFRXIYAESU-PCBIJLKTSA-N Asp-Ile-Phe Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O PYXXJFRXIYAESU-PCBIJLKTSA-N 0.000 description 1
- DWOGMPWRQQWPPF-GUBZILKMSA-N Asp-Leu-Glu Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O DWOGMPWRQQWPPF-GUBZILKMSA-N 0.000 description 1
- 201000001320 Atherosclerosis Diseases 0.000 description 1
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 description 1
- 208000028006 Corneal injury Diseases 0.000 description 1
- GSNRZJNHMVMOFV-ACZMJKKPSA-N Cys-Asp-Glu Chemical compound C(CC(=O)O)[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CS)N GSNRZJNHMVMOFV-ACZMJKKPSA-N 0.000 description 1
- ISWAQPWFWKGCAL-ACZMJKKPSA-N Cys-Cys-Glu Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(O)=O)C(O)=O ISWAQPWFWKGCAL-ACZMJKKPSA-N 0.000 description 1
- KOHBWQDSVCARMI-BWBBJGPYSA-N Cys-Cys-Thr Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CS)C(=O)N[C@@H]([C@@H](C)O)C(O)=O KOHBWQDSVCARMI-BWBBJGPYSA-N 0.000 description 1
- YUZPQIQWXLRFBW-ACZMJKKPSA-N Cys-Glu-Ala Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(O)=O YUZPQIQWXLRFBW-ACZMJKKPSA-N 0.000 description 1
- XIZWKXATMJODQW-KKUMJFAQSA-N Cys-His-Phe Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)O)NC(=O)[C@H](CC2=CN=CN2)NC(=O)[C@H](CS)N XIZWKXATMJODQW-KKUMJFAQSA-N 0.000 description 1
- DIUBVGXMXONJCF-KKUMJFAQSA-N Cys-His-Tyr Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O DIUBVGXMXONJCF-KKUMJFAQSA-N 0.000 description 1
- UVZFZTWNHOQWNK-NAKRPEOUSA-N Cys-Ile-Arg Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O UVZFZTWNHOQWNK-NAKRPEOUSA-N 0.000 description 1
- KKUVRYLJEXJSGX-MXAVVETBSA-N Cys-Ile-Phe Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)NC(=O)[C@H](CS)N KKUVRYLJEXJSGX-MXAVVETBSA-N 0.000 description 1
- OHLLDUNVMPPUMD-DCAQKATOSA-N Cys-Leu-Val Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](C(C)C)C(=O)O)NC(=O)[C@H](CS)N OHLLDUNVMPPUMD-DCAQKATOSA-N 0.000 description 1
- MKVKKORBPTUSNX-LPEHRKFASA-N Cys-Met-Pro Chemical compound CSCC[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CS)N MKVKKORBPTUSNX-LPEHRKFASA-N 0.000 description 1
- SMEYEQDCCBHTEF-FXQIFTODSA-N Cys-Pro-Ala Chemical compound [H]N[C@@H](CS)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C)C(O)=O SMEYEQDCCBHTEF-FXQIFTODSA-N 0.000 description 1
- KJJASVYBTKRYSN-FXQIFTODSA-N Cys-Pro-Asp Chemical compound C1C[C@H](N(C1)C(=O)[C@H](CS)N)C(=O)N[C@@H](CC(=O)O)C(=O)O KJJASVYBTKRYSN-FXQIFTODSA-N 0.000 description 1
- CNAMJJOZGXPDHW-IHRRRGAJSA-N Cys-Pro-Phe Chemical compound N[C@@H](CS)C(=O)N1CCC[C@H]1C(=O)N[C@@H](Cc1ccccc1)C(O)=O CNAMJJOZGXPDHW-IHRRRGAJSA-N 0.000 description 1
- FTTZLFIEUQHLHH-BWBBJGPYSA-N Cys-Thr-Cys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CS)N)O FTTZLFIEUQHLHH-BWBBJGPYSA-N 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 206010066786 Diabetic keratopathy Diseases 0.000 description 1
- 208000001351 Epiretinal Membrane Diseases 0.000 description 1
- 108700024394 Exon Proteins 0.000 description 1
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 1
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 1
- 102000009123 Fibrin Human genes 0.000 description 1
- 108010073385 Fibrin Proteins 0.000 description 1
- BWGVNKXGVNDBDI-UHFFFAOYSA-N Fibrin monomer Chemical compound CNC(=O)CNC(=O)CN BWGVNKXGVNDBDI-UHFFFAOYSA-N 0.000 description 1
- 108010049003 Fibrinogen Proteins 0.000 description 1
- 102000008946 Fibrinogen Human genes 0.000 description 1
- STHSGOZLFLFGSS-SUSMZKCASA-N Gln-Thr-Thr Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O STHSGOZLFLFGSS-SUSMZKCASA-N 0.000 description 1
- HLRLXVPRJJITSK-IFFSRLJSSA-N Gln-Thr-Val Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(O)=O HLRLXVPRJJITSK-IFFSRLJSSA-N 0.000 description 1
- DIXKFOPPGWKZLY-CIUDSAMLSA-N Glu-Arg-Asp Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(O)=O DIXKFOPPGWKZLY-CIUDSAMLSA-N 0.000 description 1
- AFODTOLGSZQDSL-PEFMBERDSA-N Glu-Asn-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CCC(=O)O)N AFODTOLGSZQDSL-PEFMBERDSA-N 0.000 description 1
- ZIYGTCDTJJCDDP-JYJNAYRXSA-N Glu-Phe-Lys Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CCC(=O)O)N ZIYGTCDTJJCDDP-JYJNAYRXSA-N 0.000 description 1
- IDEODOAVGCMUQV-GUBZILKMSA-N Glu-Ser-Leu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(O)=O IDEODOAVGCMUQV-GUBZILKMSA-N 0.000 description 1
- DXMOIVCNJIJQSC-QEJZJMRPSA-N Glu-Trp-Ser Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CO)C(=O)O)NC(=O)[C@H](CCC(=O)O)N DXMOIVCNJIJQSC-QEJZJMRPSA-N 0.000 description 1
- MIWJDJAMMKHUAR-ZVZYQTTQSA-N Glu-Trp-Val Chemical compound CC(C)[C@@H](C(=O)O)NC(=O)[C@H](CC1=CNC2=CC=CC=C21)NC(=O)[C@H](CCC(=O)O)N MIWJDJAMMKHUAR-ZVZYQTTQSA-N 0.000 description 1
- QXPRJQPCFXMCIY-NKWVEPMBSA-N Gly-Ala-Pro Chemical compound C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)CN QXPRJQPCFXMCIY-NKWVEPMBSA-N 0.000 description 1
- XQHSBNVACKQWAV-WHFBIAKZSA-N Gly-Asp-Asn Chemical compound [H]NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O XQHSBNVACKQWAV-WHFBIAKZSA-N 0.000 description 1
- ZRZILYKEJBMFHY-BQBZGAKWSA-N Gly-Asp-Met Chemical compound CSCC[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)CN ZRZILYKEJBMFHY-BQBZGAKWSA-N 0.000 description 1
- NMROINAYXCACKF-WHFBIAKZSA-N Gly-Cys-Cys Chemical compound NCC(=O)N[C@@H](CS)C(=O)N[C@@H](CS)C(O)=O NMROINAYXCACKF-WHFBIAKZSA-N 0.000 description 1
- JSNNHGHYGYMVCK-XVKPBYJWSA-N Gly-Glu-Val Chemical compound [H]NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O JSNNHGHYGYMVCK-XVKPBYJWSA-N 0.000 description 1
- 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
- TVUWMSBGMVAHSJ-KBPBESRZSA-N Gly-Leu-Phe Chemical compound NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 TVUWMSBGMVAHSJ-KBPBESRZSA-N 0.000 description 1
- VEPBEGNDJYANCF-QWRGUYRKSA-N Gly-Lys-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CCCCN VEPBEGNDJYANCF-QWRGUYRKSA-N 0.000 description 1
- HHRODZSXDXMUHS-LURJTMIESA-N Gly-Met-Gly Chemical compound CSCC[C@H](NC(=O)C[NH3+])C(=O)NCC([O-])=O HHRODZSXDXMUHS-LURJTMIESA-N 0.000 description 1
- QSQXZZCGPXQBPP-BQBZGAKWSA-N Gly-Pro-Cys Chemical compound C1C[C@H](N(C1)C(=O)CN)C(=O)N[C@@H](CS)C(=O)O QSQXZZCGPXQBPP-BQBZGAKWSA-N 0.000 description 1
- ABPRMMYHROQBLY-NKWVEPMBSA-N Gly-Ser-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CO)NC(=O)CN)C(=O)O ABPRMMYHROQBLY-NKWVEPMBSA-N 0.000 description 1
- SBVMXEZQJVUARN-XPUUQOCRSA-N Gly-Val-Ser Chemical compound NCC(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(O)=O SBVMXEZQJVUARN-XPUUQOCRSA-N 0.000 description 1
- 206010018473 Glycosuria Diseases 0.000 description 1
- 101001095308 Homo sapiens Periostin Proteins 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- UGQMRVRMYYASKQ-UHFFFAOYSA-N Hypoxanthine nucleoside Natural products OC1C(O)C(CO)OC1N1C(NC=NC2=O)=C2N=C1 UGQMRVRMYYASKQ-UHFFFAOYSA-N 0.000 description 1
- TZCGZYWNIDZZMR-UHFFFAOYSA-N Ile-Arg-Ala Natural products CCC(C)C(N)C(=O)NC(C(=O)NC(C)C(O)=O)CCCN=C(N)N TZCGZYWNIDZZMR-UHFFFAOYSA-N 0.000 description 1
- VGSPNSSCMOHRRR-BJDJZHNGSA-N Ile-Ser-Lys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)O)N VGSPNSSCMOHRRR-BJDJZHNGSA-N 0.000 description 1
- RQJUKVXWAKJDBW-SVSWQMSJSA-N Ile-Ser-Thr Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(=O)O)N RQJUKVXWAKJDBW-SVSWQMSJSA-N 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 108010065920 Insulin Lispro Proteins 0.000 description 1
- HGCNKOLVKRAVHD-UHFFFAOYSA-N L-Met-L-Phe Natural products CSCCC(N)C(=O)NC(C(O)=O)CC1=CC=CC=C1 HGCNKOLVKRAVHD-UHFFFAOYSA-N 0.000 description 1
- LHSGPCFBGJHPCY-UHFFFAOYSA-N L-leucine-L-tyrosine Natural products CC(C)CC(N)C(=O)NC(C(O)=O)CC1=CC=C(O)C=C1 LHSGPCFBGJHPCY-UHFFFAOYSA-N 0.000 description 1
- FOEHRHOBWFQSNW-KATARQTJSA-N Leu-Cys-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CS)NC(=O)[C@H](CC(C)C)N)O FOEHRHOBWFQSNW-KATARQTJSA-N 0.000 description 1
- HFBCHNRFRYLZNV-GUBZILKMSA-N Leu-Glu-Asp Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O HFBCHNRFRYLZNV-GUBZILKMSA-N 0.000 description 1
- KGCLIYGPQXUNLO-IUCAKERBSA-N Leu-Gly-Glu Chemical compound CC(C)C[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCC(O)=O KGCLIYGPQXUNLO-IUCAKERBSA-N 0.000 description 1
- JDBQSGMJBMPNFT-AVGNSLFASA-N Leu-Pro-Val Chemical compound CC(C)C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C(C)C)C(O)=O JDBQSGMJBMPNFT-AVGNSLFASA-N 0.000 description 1
- NNCDAORZCMPZPX-GUBZILKMSA-N Lys-Gln-Ser Chemical compound C(CCN)C[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CO)C(=O)O)N NNCDAORZCMPZPX-GUBZILKMSA-N 0.000 description 1
- IVFUVMSKSFSFBT-NHCYSSNCSA-N Lys-Ile-Gly Chemical compound OC(=O)CNC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@@H](N)CCCCN IVFUVMSKSFSFBT-NHCYSSNCSA-N 0.000 description 1
- MTBBHUKKPWKXBT-ULQDDVLXSA-N Lys-Met-Tyr Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O MTBBHUKKPWKXBT-ULQDDVLXSA-N 0.000 description 1
- TVOOGUNBIWAURO-KATARQTJSA-N Lys-Thr-Cys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CCCCN)N)O TVOOGUNBIWAURO-KATARQTJSA-N 0.000 description 1
- YFQSSOAGMZGXFT-MEYUZBJRSA-N Lys-Thr-Tyr Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O YFQSSOAGMZGXFT-MEYUZBJRSA-N 0.000 description 1
- XATKLFSXFINPSB-JYJNAYRXSA-N Lys-Tyr-Gln Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCC(N)=O)C(O)=O XATKLFSXFINPSB-JYJNAYRXSA-N 0.000 description 1
- FVKRQMQQFGBXHV-QXEWZRGKSA-N Met-Asp-Val Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O FVKRQMQQFGBXHV-QXEWZRGKSA-N 0.000 description 1
- HAQLBBVZAGMESV-IHRRRGAJSA-N Met-Lys-Lys Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(O)=O HAQLBBVZAGMESV-IHRRRGAJSA-N 0.000 description 1
- YBAFDPFAUTYYRW-UHFFFAOYSA-N N-L-alpha-glutamyl-L-leucine Natural products CC(C)CC(C(O)=O)NC(=O)C(N)CCC(O)=O YBAFDPFAUTYYRW-UHFFFAOYSA-N 0.000 description 1
- KZNQNBZMBZJQJO-UHFFFAOYSA-N N-glycyl-L-proline Natural products NCC(=O)N1CCCC1C(O)=O KZNQNBZMBZJQJO-UHFFFAOYSA-N 0.000 description 1
- 208000037273 Pathologic Processes Diseases 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- ALHULIGNEXGFRM-QWRGUYRKSA-N Phe-Cys-Gly Chemical compound OC(=O)CNC(=O)[C@H](CS)NC(=O)[C@@H](N)CC1=CC=CC=C1 ALHULIGNEXGFRM-QWRGUYRKSA-N 0.000 description 1
- KJJROSNFBRWPHS-JYJNAYRXSA-N Phe-Glu-Leu Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O KJJROSNFBRWPHS-JYJNAYRXSA-N 0.000 description 1
- WEMYTDDMDBLPMI-DKIMLUQUSA-N Phe-Ile-Lys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)N WEMYTDDMDBLPMI-DKIMLUQUSA-N 0.000 description 1
- LNLNHXIQPGKRJQ-SRVKXCTJSA-N Pro-Arg-Arg Chemical compound NC(N)=NCCC[C@@H](C(O)=O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@@H]1CCCN1 LNLNHXIQPGKRJQ-SRVKXCTJSA-N 0.000 description 1
- YFNOUBWUIIJQHF-LPEHRKFASA-N Pro-Asp-Pro Chemical compound C1C[C@H](NC1)C(=O)N[C@@H](CC(=O)O)C(=O)N2CCC[C@@H]2C(=O)O YFNOUBWUIIJQHF-LPEHRKFASA-N 0.000 description 1
- QXNSKJLSLYCTMT-FXQIFTODSA-N Pro-Cys-Asp Chemical compound C1C[C@H](NC1)C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(=O)O)C(=O)O QXNSKJLSLYCTMT-FXQIFTODSA-N 0.000 description 1
- UIMCLYYSUCIUJM-UWVGGRQHSA-N Pro-Gly-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H]1CCCN1 UIMCLYYSUCIUJM-UWVGGRQHSA-N 0.000 description 1
- LCUOTSLIVGSGAU-AVGNSLFASA-N Pro-His-Arg Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O LCUOTSLIVGSGAU-AVGNSLFASA-N 0.000 description 1
- BAKAHWWRCCUDAF-IHRRRGAJSA-N Pro-His-Lys Chemical compound C([C@@H](C(=O)N[C@@H](CCCCN)C(O)=O)NC(=O)[C@H]1NCCC1)C1=CN=CN1 BAKAHWWRCCUDAF-IHRRRGAJSA-N 0.000 description 1
- LXLFEIHKWGHJJB-XUXIUFHCSA-N Pro-Ile-Lys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@@H]1CCCN1 LXLFEIHKWGHJJB-XUXIUFHCSA-N 0.000 description 1
- ZLXKLMHAMDENIO-DCAQKATOSA-N Pro-Lys-Asp Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(O)=O ZLXKLMHAMDENIO-DCAQKATOSA-N 0.000 description 1
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 206010038848 Retinal detachment Diseases 0.000 description 1
- 208000017442 Retinal disease Diseases 0.000 description 1
- 206010038923 Retinopathy Diseases 0.000 description 1
- 206010070834 Sensitisation Diseases 0.000 description 1
- SVWQEIRZHHNBIO-WHFBIAKZSA-N Ser-Gly-Cys Chemical compound [H]N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CS)C(O)=O SVWQEIRZHHNBIO-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
- FZEUTKVQGMVGHW-AVGNSLFASA-N Ser-Phe-Gln Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCC(N)=O)C(O)=O FZEUTKVQGMVGHW-AVGNSLFASA-N 0.000 description 1
- PJIQEIFXZPCWOJ-FXQIFTODSA-N Ser-Pro-Asp Chemical compound [H]N[C@@H](CO)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(O)=O)C(O)=O PJIQEIFXZPCWOJ-FXQIFTODSA-N 0.000 description 1
- VFWQQZMRKFOGLE-ZLUOBGJFSA-N Ser-Ser-Cys Chemical compound C([C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CS)C(=O)O)N)O VFWQQZMRKFOGLE-ZLUOBGJFSA-N 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
- 201000009594 Systemic Scleroderma Diseases 0.000 description 1
- 206010042953 Systemic sclerosis Diseases 0.000 description 1
- QWMPARMKIDVBLV-VZFHVOOUSA-N Thr-Cys-Ala Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](CS)C(=O)N[C@@H](C)C(O)=O QWMPARMKIDVBLV-VZFHVOOUSA-N 0.000 description 1
- QFEYTTHKPSOFLV-OSUNSFLBSA-N Thr-Met-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CCSC)NC(=O)[C@H]([C@@H](C)O)N QFEYTTHKPSOFLV-OSUNSFLBSA-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
- GFRIEEKFXOVPIR-RHYQMDGZSA-N Thr-Pro-Lys Chemical compound C[C@@H](O)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCCN)C(O)=O GFRIEEKFXOVPIR-RHYQMDGZSA-N 0.000 description 1
- XZUBGOYOGDRYFC-XGEHTFHBSA-N Thr-Ser-Met Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCSC)C(O)=O XZUBGOYOGDRYFC-XGEHTFHBSA-N 0.000 description 1
- COYHRQWNJDJCNA-NUJDXYNKSA-N Thr-Thr-Thr Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O COYHRQWNJDJCNA-NUJDXYNKSA-N 0.000 description 1
- 108090000190 Thrombin Proteins 0.000 description 1
- GPLTZEMVOCZVAV-UFYCRDLUSA-N Tyr-Tyr-Arg Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O)C1=CC=C(O)C=C1 GPLTZEMVOCZVAV-UFYCRDLUSA-N 0.000 description 1
- CVUDMNSZAIZFAE-TUAOUCFPSA-N Val-Arg-Pro Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N1CCC[C@@H]1C(=O)O)N CVUDMNSZAIZFAE-TUAOUCFPSA-N 0.000 description 1
- CVUDMNSZAIZFAE-UHFFFAOYSA-N Val-Arg-Pro Natural products NC(N)=NCCCC(NC(=O)C(N)C(C)C)C(=O)N1CCCC1C(O)=O CVUDMNSZAIZFAE-UHFFFAOYSA-N 0.000 description 1
- BEGDZYNDCNEGJZ-XVKPBYJWSA-N Val-Gly-Gln Chemical compound CC(C)[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCC(N)=O BEGDZYNDCNEGJZ-XVKPBYJWSA-N 0.000 description 1
- YTPLVNUZZOBFFC-SCZZXKLOSA-N Val-Gly-Pro Chemical compound CC(C)[C@H](N)C(=O)NCC(=O)N1CCC[C@@H]1C(O)=O YTPLVNUZZOBFFC-SCZZXKLOSA-N 0.000 description 1
- ZRSZTKTVPNSUNA-IHRRRGAJSA-N Val-Lys-Leu Chemical compound CC(C)C[C@H](NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)C(C)C)C(O)=O ZRSZTKTVPNSUNA-IHRRRGAJSA-N 0.000 description 1
- JXCOEPXCBVCTRD-JYJNAYRXSA-N Val-Tyr-Arg Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)N JXCOEPXCBVCTRD-JYJNAYRXSA-N 0.000 description 1
- 208000036866 Vitreoretinopathy Diseases 0.000 description 1
- 210000000683 abdominal cavity Anatomy 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 108010047495 alanylglycine Proteins 0.000 description 1
- 150000001335 aliphatic alkanes Chemical class 0.000 description 1
- KOSRFJWDECSPRO-UHFFFAOYSA-N alpha-L-glutamyl-L-glutamic acid Natural products OC(=O)CCC(N)C(=O)NC(CCC(O)=O)C(O)=O KOSRFJWDECSPRO-UHFFFAOYSA-N 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 229960003896 aminopterin Drugs 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000033115 angiogenesis Effects 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 229940125644 antibody drug Drugs 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 108010093581 aspartyl-proline Proteins 0.000 description 1
- 108010047857 aspartylglycine Proteins 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 1
- 238000003766 bioinformatics method Methods 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 208000002352 blister Diseases 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000004709 cell invasion Effects 0.000 description 1
- 230000012292 cell migration Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 239000003638 chemical reducing agent Substances 0.000 description 1
- 230000003399 chemotactic effect Effects 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 230000004087 circulation Effects 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 108010016616 cysteinylglycine Proteins 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 230000029578 entry into host Effects 0.000 description 1
- 210000002744 extracellular matrix Anatomy 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 208000030533 eye disease Diseases 0.000 description 1
- 229950003499 fibrin Drugs 0.000 description 1
- 229940012952 fibrinogen Drugs 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 108010055341 glutamyl-glutamic acid Proteins 0.000 description 1
- VPZXBVLAVMBEQI-UHFFFAOYSA-N glycyl-DL-alpha-alanine Natural products OC(=O)C(C)NC(=O)CN VPZXBVLAVMBEQI-UHFFFAOYSA-N 0.000 description 1
- 108010077515 glycylproline Proteins 0.000 description 1
- 108010037850 glycylvaline Proteins 0.000 description 1
- 210000002216 heart Anatomy 0.000 description 1
- 102000048431 human POSTN Human genes 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 210000004969 inflammatory cell Anatomy 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 230000002427 irreversible effect Effects 0.000 description 1
- 230000000302 ischemic effect Effects 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 108010053037 kyotorphin Proteins 0.000 description 1
- 108010057821 leucylproline Proteins 0.000 description 1
- 108010012058 leucyltyrosine Proteins 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 108010064235 lysylglycine Proteins 0.000 description 1
- 108010054155 lysyllysine Proteins 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- FBOZXECLQNJBKD-UHFFFAOYSA-N methotrexate Chemical compound C=1N=C2N=C(N)N=C(N)C2=NC=1CN(C)C1=CC=C(C(=O)NC(CCC(O)=O)C(O)=O)C=C1 FBOZXECLQNJBKD-UHFFFAOYSA-N 0.000 description 1
- 230000000116 mitigating effect Effects 0.000 description 1
- 239000003226 mitogen Substances 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 239000013618 particulate matter Substances 0.000 description 1
- 230000009054 pathological process Effects 0.000 description 1
- 108010070409 phenylalanyl-glycyl-glycine Proteins 0.000 description 1
- 108010089198 phenylalanyl-prolyl-arginine Proteins 0.000 description 1
- 108010012581 phenylalanylglutamate Proteins 0.000 description 1
- 210000002826 placenta Anatomy 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 1
- 238000003825 pressing Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 108010090894 prolylleucine Proteins 0.000 description 1
- 208000037922 refractory disease Diseases 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000004264 retinal detachment Effects 0.000 description 1
- 230000002207 retinal effect Effects 0.000 description 1
- 238000005185 salting out Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 231100000241 scar Toxicity 0.000 description 1
- 239000006152 selective media Substances 0.000 description 1
- 230000008313 sensitization Effects 0.000 description 1
- 108010071207 serylmethionine Proteins 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 238000004611 spectroscopical analysis Methods 0.000 description 1
- 230000007480 spreading Effects 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
- 229960004072 thrombin Drugs 0.000 description 1
- 230000000451 tissue damage Effects 0.000 description 1
- 231100000827 tissue damage Toxicity 0.000 description 1
- 230000003827 upregulation Effects 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
- 230000037314 wound repair Effects 0.000 description 1
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Peptides Or Proteins (AREA)
Abstract
The present invention provides a kind of with the monoclonal antibody and its preparation method and application for inhibiting the effect of vitreous-body-retina fibrosis lesion.The monoclonal antibody is anti-human CTGF monoclonal's antibody, and the antibody fragment of anti-human CTGF monoclonal's antibody includes single chain variable fragment, Fab, Fab ', F (ab ') 2 or nano antibody.Monoclonal antibody of the invention can be specifically bound with the CTGF in blood in human body, body fluid, to have the function that inhibit CTGF that cell is promoted to form micro-pipe, to prevent and treat vitreous-body-retina fibrosis lesion, suitable for preparing the monoclonal antibody drug for the treatment of vitreous-body-retina fibrosis lesion.
Description
Technical field
The present invention relates to monoclonal antibody technique fields, more particularly to one kind to have inhibition vitreous-body-retina fibrotic disease
Monoclonal antibody being changed into and its preparation method and application.
Background technique
Connective Tissue Growth Factor (Connective tissue growth factor, CTGF) is promoted after tissue damage
The important cytokine formed into fibrotic scar, can express in the histoorgans such as the heart, brain, kidney, lung, liver and placenta.
In No. 6 chromosome long arm, including 5 exons and 4 intrones, encoding albumen is by 349 for the mankind CTGF assignment of genes gene mapping
The molecular mass of a Amino acid profile is the secretion peptide rich in cysteine of 36~38kD, with transforming growth factor-β (TGF-
β) there is close relationship, can be considered the downstream reaction element of TGF-β.In cicatrization, organ fibrosis, Atherosclerosis
In the diseases such as change, systemic sclerosis, wound repair and some good, malignant tumours, CTGF has different degrees of expression up-regulation.
In addition, the pathologic processes such as the generation of CTGF and fibrotic disease, development have close relationship, it has also become alternative
The new targeted cellular elements of TGF-β treatment fibrotic disease.The difference of CTGF site of action, the biological effect of performance is not yet
Together, it is mainly shown as and cell mitogen and hyperplasia, chemotactic cell, inducing cell adherency is promoted to promote the conjunction of extracellular matrix
At.CTGF can also adjust angiogenesis, induce cell apoptosis.
Ocular tissue's fibrosis is relatively conventional in ophthalmology disease, glaucoma filtration operation, eye traumas, diabetic keratopathy view
Also the fibrosis of tissue is directed to after film lesion and vitrectomy.Due to the particularity of eye structure, especially
Operation and wound cause blood-eye barrier to destroy, corneal injury, and vasopermeability increases, and make fibrinogen, inflammatory cell, at fibre
It ties up cell migration to enter intraocularly, is then changed into fibrin under thrombin action.Ocular tissue caused by various factors damages is fine
Dimensionization causes irreversible side effect to vision restoration, so inhibiting the fibrosis of injury tissue, mitigating cicatrization is to guarantee
The key of eyesight and global problem.
Proliferative vitreoretinopathy (proliferative vitreoretinopathy, PVR) is a kind of because of inflammation
Disease, damage, excessive prosthetic disease caused by ischemic, disease incidence reaches 10% in retinal detachment patients, and essence is
Intraocular fibrosis.In ophthalmology, it further includes diabetic retina in addition to PVR that fibrotic disease, which is a kind of ophthalmology refractory disease,
Lesion proliferation period, the neovascular membranes of retina, preretinal membrane, glaucoma filtering operation avascular filtering bleb fibrosis etc., these are all
It is that disease incidence is high, endangers serious blinding eye disease.However, there are many complication and secondary work for existing clinical commonly used drug
With other kinds for the treatment of method some is only limitted to animal experiment stage, still up for further clinical research.
Therefore, it needs to find and a kind of more useful resists intraocular fibrosis approach.
Summary of the invention
The purpose of the present invention is to provide a kind of monoclonal with inhibition vitreous-body-retina fibrosis lesion effect is anti-
Body and its preparation method and application, the monoclonal antibody can effectively inhibit the vitreous-body-retina fiber due to caused by CTGF
Change lesion.
An aspect of of the present present invention provides a kind of monoclonal antibody with inhibition vitreous-body-retina fibrosis lesion effect,
The monoclonal antibody be anti-human CTGF monoclonal's antibody, the antibody fragment of anti-human CTGF monoclonal's antibody include it is single-stranded can
Become area's segment, Fab, Fab ', F (ab ') 2 or nano antibody.
Further, the antibody fragment merges or is modified agent modification with other peptide or protein matter.
It is anti-that another aspect of the present invention provides a kind of monoclonal with inhibition vitreous-body-retina fibrosis lesion effect
The preparation method of body, comprising: obtain nucleic acid sequence recombined human CTGF antigen protein as shown in SEQ ID NO:1；With described heavy
Mouse is immunized in group people CTGF antigen protein, and separation obtains splenocyte in the mouse after being immunized；By the mouse boosting cell with
Homology myeloma cell mixes, and carries out film fusion using PEG, forms hybridoma；Screen the hybridization of successfully film fusion
Oncocyte carries out in-vitro multiplication culture, hybridoma is inoculated in mouse peritoneal, from the mouse after inoculation after the predetermined time
The monoclonal antibody is obtained in ascites.
Further, the step of obtaining the monoclonal antibody from the ascites of the mouse after inoculation includes: to the abdomen
Water is slightly purified；It dialyses to the product slightly purified；It is purified using product of the HPLC method to the dialysis,
In, the purifying uses anionic exchange medium, and using mobile phase A and Mobile phase B as eluent, flow velocity is 1~3mL/min, benefit
It is eluted with gradient elution mode, 35~40 DEG C of column temperature, Detection wavelength 280nm；The mobile phase A is trihydroxy methyl amino first
Alkane buffer, the Mobile phase B are the mixed solution of TRIS buffer and sodium chloride.
Further, the step of mouse being immunized with the recombined human CTGF antigen protein includes: in the recombined human CTGF
Freund's complete adjuvant is added in antigen protein, first immunisation injection is carried out to mouse；In the recombined human CTGF antigen protein
Incomplete Freund's adjuvant is added, booster immunization injection is carried out to the mouse after first immunisation.
Further, by 1 × 108A mouse boosting cell and 2~3 × 107A homology myeloma cell mixes.
Application of the monoclonal antibody provided by the invention in preparation treatment vitreous-body-retina fibrosis lesion drug.
Further, the vitreous-body-retina fibrosis lesion includes: glaucoma filtration operation, eye traumas, glycosuria
Caused vitreous-body-retina fibrosis lesion after characteristic of disease retinopathy and vitrectomy.
Compared with prior art, the beneficial effects of the present invention are:
It is provided by the invention excellent with inhibiting the monoclonal antibody of vitreous-body-retina fibrosis lesion effect to have
It in conjunction with activity and/or neutralization activity, can specifically bind with the CTGF in blood in human body, body fluid, to remove CTGF, press down
CTGF processed promotes cell to form micro-pipe, and then the process of barrier fibers, protects retinal tissue, reaches prevention and treatment hyperplasia
Property vitreous-body-retina fibrosis lesion effect, the monoclonal suitable for preparing treatment vitreous-body-retina fibrosis lesion is anti-
Body drug.
Detailed description of the invention
Fig. 1 is the flow chart that the embodiment of the present invention prepares anti-human CTGF monoclonal's antibody.
Fig. 2A is the electrophoresis result figure of the expression and purification of CTGF recombinant protein；
Wherein, FT is to flow through liquid component；W1-W3 is low concentration elution component；MW is protein Marker；E1-
E9 is purpose PROTEIN C TGF difference appearance time electrophoresis result.
Fig. 2 B is the quantitative electrophoresis result of 2 μ g destination protein CTGF.
Fig. 3 is the monoclonal antibody reduction SDS-PAGE electrophoresis result of purifying；
Wherein, M is protein Marker；1 is anti-human CTGF monoclonal's antibody.
Fig. 4 is the WB result of anti-human CTGF monoclonal's antibody and antigens c TGF specific binding.
Fig. 5 is invasive ability figure of anti-human CTGF monoclonal's antibody to Rat Smooth Muscle；
Wherein, A is normal cell culture, and cell number is the 1605/visual field；B is that IgG, cell is added in lower layer's culture medium
Number is the 998/visual field；C is that anti-human CTGF monoclonal's antibody 1 is added in lower layer's culture medium, and cell number is the 727/visual field；D is
Anti-human CTGF monoclonal's antibody 2 is added in lower layer's culture medium, cell number is the 562/visual field.
Specific embodiment
Now, example embodiment is more fully described with reference to the accompanying drawings, wherein some exemplary embodiments are shown in the accompanying drawings
Out.
The embodiment of the present invention provides a kind of monoclonal antibody with inhibition vitreous-body-retina fibrosis lesion effect,
Its specific antigen is nucleic acid sequence as shown in SEQ ID NO:1, amino acid sequence people CTGF as shown in SEQ ID NO:2.
The monoclonal antibody is anti-human CTGF monoclonal's antibody, the antibody fragment packet of anti-human CTGF monoclonal's antibody
Include single chain variable fragment, Fab, Fab ', F (ab ') 2 or nano antibody.
The antibody fragment merges or is modified agent modification with other peptide or protein matter.
Have the monoclonal for inhibiting the effect of vitreous-body-retina fibrosis lesion anti-below with reference to Fig. 1 to Fig. 5 detailed description
The preparation method of body.
Referring to Fig.1, the embodiment of the present invention provides a kind of preparation method of anti-human CTGF monoclonal's antibody, comprising:
In step S10, nucleic acid sequence recombined human CTGF antigen protein as shown in SEQ ID NO:1 is obtained.
As an example, the preparation method of recombined human CTGF antigen protein is as follows:
1, by bioinformatics method, retrieval obtains CTGF nucleic acid sequence such as SEQ ID NO:1 in ncbi database
It is shown.According to this nucleic acid sequence information, the DNA of artificial synthesized CTGF.
2, using the DNA of the artificial synthesized CTGF as template, PCR amplification is carried out.
PCR primer is as follows:
P1:GAATTCATGACCGCCGCCAGTATG
P2:GCGGCCGCTCATGCCATGTCTCCGTA
PCR system is as follows: 5 μ L, dNTP Mixture of Ex Taq (5U/ μ L) 0.25 μ L, 10 × Ex Taq Buffer
(2.5mM) 4 μ L, Template 1 μ L, upstream primer P1 (10 μM) 1 μ L, downstream primer P2 (10 μM) 1 μ L, ddH2O complements to 50
μL。
PCR amplification condition: 93 DEG C of denaturation temperature, 55 DEG C of annealing temperature, 72 DEG C of elongating temperature, 30~40 circulations, the time
It is 2~3 hours.
3, obtained PCR product is linked on pATX2 with EcoR/Not I double digestion method, while 6*His label is added,
Construction recombination plasmid.
4, it will be cultivated after the Transfected Recombinant Plasmid HEK293 cell, sample is collected after six days and carry out SDS-PAGE
Electrophoresis detection and protein immunoblotting (western blotting) detection, inspection result obtain expression strain.
5, strain is expressed described in mass propgation and obtains recombined human CTGF antigen protein through affinitive layer purification.
The recombined human CTGF antigen protein of acquisition is made into PAGE gel electroresis appraisal, electrophoresis result such as Fig. 2A and Fig. 2 B
It is shown.E1-E9 is purpose PROTEIN C TGF antigen protein difference appearance time electrophoresis result in Fig. 2A, and Fig. 2 B indicates that applied sample amount is 2 μ
When g, the quantitative electrophoresis result of destination protein CTGF.By the visible indistinct band of Fig. 2 B, concentration reaches requirement.
General strong biological (Wuhan) Science and Technology Ltd. is sent to carry out genetic test the recombined human CTGF antigen protein of acquisition,
Nucleic acid sequence is as shown in SEQ ID NO:1, and amino acid sequence is as shown in SEQ ID NO:2, it can be seen that, successfully synthesize recombination
People's CTGF antigen protein.
It should be appreciated that the molecular biology method using this field routine obtains people CTGF antigen protein, the present invention couple
This is not construed as limiting.
In step S20, mouse is immunized with the recombined human CTGF antigen protein of acquisition, and separate and obtain in the mouse after being immunized
Obtain splenocyte.
As an example, selecting 6~8 week old Balb/c mouse is immune animal, it is immune to carry out recombined human CTGF antigen protein
Injection stimulates corresponding bone-marrow-derived lymphocyte activation, is proliferated and is divided into sensitization bone-marrow-derived lymphocyte.
Preferably, 8 week old Balb/c mouse are immunized with the recombined human periostin antigen protein of acquisition.
In one embodiment, the step of mouse being immunized with the recombined human CTGF antigen protein includes: in the recombination
Freund's complete adjuvant is added in people's CTGF antigen protein, first immunisation injection is carried out to mouse；In the recombined human CTGF antigen
Incomplete Freund's adjuvant is added in albumen, booster immunization injection is carried out to the mouse after first immunisation.
As an example, Freund's complete adjuvant is added in recombined human CTGF antigen protein, by every 10~100 μ g recombined human of mouse
The dosage of CTGF antigen protein carries out first immunisation, and the nape of the neck for being injected in mouse is subcutaneous (multiple spot) or intraperitoneal.
Booster immunization uses the recombined human CTGF antigen protein of one half-value dose of first immunisation, and incomplete Freund's adjuvant is added,
Booster immunization is carried out, the nape of the neck for being injected in mouse is subcutaneous (multiple spot) or intraperitoneal.It takes within 7~10 days blood to survey potency after immune, adds
After being immunized 3~4 times by force, separation obtains mouse boosting cell.
Preferably, first immunisation is carried out by the dosage of every 50 μ g recombined human CTGF antigen protein of mouse, is injected in mouse
Subcutaneous 4 points of the nape of the neck；Booster immunization is carried out by the dosage of every 25 μ g recombined human CTGF antigen protein of mouse, is injected in small
Subcutaneous 4 points of the nape of the neck of mouse.Blood is taken within 8 days to survey potency after immune, after booster immunization 4 times, separation obtains mouse boosting cell.
In step S30, the mouse boosting cell is mixed with homology myeloma cell, is utilized PEG (polyethylene glycol)
Film fusion is carried out, hybridoma is formed.
As an example, the mouse boosting cell and homology myeloma for obtaining separation are thin using hybridoma cell fusion technology
Born of the same parents mix, and carry out film fusion using PEG, form hybridoma.
Preferably, by 1 × 108A mouse boosting cell and 2~3 × 107A homology myeloma cell mixes.
As an example, pressing 1 × 108A mouse boosting cell and 3 × 107A homology myeloma cell mixes.
It should be appreciated that can be anti-using hybridoma cell fusion technology and Cloning Technology the preparation secretion of this field routine
The hybridoma cell strain of purpose antigen CTGF.
In step S40, the hybridoma of successfully film fusion is screened, in-vitro multiplication culture is carried out, it is inscribed in mouse peritoneal
Kind hybridoma, obtains the monoclonal antibody from the ascites of the mouse after inoculation after the predetermined time.
Preferably, it is greater than 1.5 through ELISA detection OD495, is successfully the hybridoma of film fusion.
As an example, the screening of hybridoma positive cell and cloning: in HAT (H-Hypoxanthine hypoxanthine, A-
Aminopterin methopterin, T-Thymidine thymidine) the miscellaneous of successfully film fusion is screened on selective medium
Friendship oncocyte, in-vitro multiplication culture, and in time to mouse (non-immune conventional mouse, such as 8 week old Balb/c mouse) abdominal cavity
Interior inoculation hybridoma prepares ascites, and ascites is extracted after about 1~2 week, obtains a large amount of thick monoclonal antibodies.Here, extraction
Ascites is the mouse ascites containing a large amount of thick monoclonal antibodies.
In addition, being purified to thick monoclonal antibody.
Preferably, the step of obtaining the monoclonal antibody from the ascites of the mouse after inoculation can include: to the abdomen
Water is slightly purified；It dialyses to the product slightly purified；It is purified using product of the HPLC method to the dialysis.
As an example, carrying out salt to the mouse ascites containing monoclonal antibody using saturated ammonium sulfate solution based on salting out method
Analysis.
Specifically, the mouse ascites stoste containing a large amount of thick monoclonal antibodies is subjected to first time low-temperature centrifugation processing (4
DEG C, 10000rpm, 15min), to remove cell residue and particulate matter etc., supernatant obtained by first time low-temperature centrifugation is used
0.02mol/L, pH value be 7.2 phosphate buffer be diluted to 5 times (that is, the first time low-temperature centrifugation obtained by supernatant with
The volume ratio of the phosphate buffer is 1:4, and the total volume after being diluted to 5 times is denoted as sample volume) after, in ice bath stirring
Under, saturated ammonium sulfate solution is added to final concentration of 50% (v/v) (that is, the sample volume and the saturated ammonium sulfate solution
Volume ratio be 1:1), saltout in 4 DEG C 4 hours, and carry out second (4 DEG C, 10000rpm, 15min) of low-temperature centrifugation processing, abandon
Supernatant obtains solids.Then, 0.02mol/L, pH value is used to dissolve second of low-temperature centrifugation institute for 7.2 phosphate buffer
Solids is obtained, the volume of the phosphate buffer is equal with the sample volume, and under ice bath stirring, saturation sulphur is added dropwise
Acid ammonium solution (here, the initial value of the concentration of saturated ammonium sulfate solution is 1g/mL) extremely final concentration of 33.33% (v/v) (that is,
The volume ratio of dissolved total volume and the saturated ammonium sulfate solution is 2:1), it saltouts in 4 DEG C overnight, and it is low to carry out third time
(4 DEG C, 10000rpm, 15min) processing of temperature centrifugation, abandon supernatant and obtain solids, third time low-temperature centrifugation obtained solid object is made
For the thick purification sample of gained of saltouing, i.e., the product slightly purified.
As an example, being dialysed using TRIS buffer to the product slightly purified.
Specifically, gained after using 0.025mol/L, pH value to saltout for 7.2 TRIS buffer dissolution
Thick purification sample, the volume of the TRIS buffer are equal with the sample volume；Then it is using concentration
During which 0.025mol/L, the TRIS buffer that pH value is 7.2 need to change 3 three hydroxyls in 4 DEG C of dialysed overnights
Aminomethane buffer, molecular cut off 150KD, to obtain the product of dialysis.
As an example, utilizing HPLC method (High Performance Liquid Chromatography, high-efficient liquid phase color
Spectrometry) product of dialysis is purified.The purifying uses anionic exchange medium, is elution with mobile phase A and Mobile phase B
Liquid, flow velocity are 1~3mL/min, are eluted using gradient elution mode, 35~40 DEG C of column temperature, Detection wavelength 280nm；It is described
Mobile phase A is that pH value is 6.5~8.5, and concentration is the buffer of the trishydroxymethylaminomethane of 10~50mmol/L；The flowing
Phase B is that pH value is 6.5~8.5, the final concentration of 10~50mmol/L of trishydroxymethylaminomethane and sodium chloride final concentration of 0.5~
The mixed solution of 1mol/L.
Preferably, using Thermo Propac-wax-10 chromatographic column, the granularity of filler is about 10 μm, chromatographic column specification
For 250mm × 4.6mm, select mobile phase A and Mobile phase B be eluent, flow velocity 1mL/min, using gradient elution mode into
Row elution, 40 DEG C of column temperature, Detection wavelength 280nm；The mobile phase A is that pH value is 7, and concentration is the trihydroxy methyl of 30mmol/L
Aminomethane buffer solution；The Mobile phase B is that pH value is 7, the final concentration of 30mmol/L of TRIS buffer and
The mixed solution of the final concentration of 1mol/L of sodium chloride.Chromatographic column is connected, chromatographic column is balanced with mobile phase A.Loading, loading
Gradient program is executed afterwards to isolate and purify sample, carries out gradient elution by the elution program of table 1, collecting retention time is 22
~24 minutes eluting peaks obtain anti-human CTGF monoclonal's antibody of purifying.Yield is 85.2%, purity 97.4%.
Table 1: gradient elution program
It should be appreciated that can also be purified using the purification process of this field routine to monoclonal antibody, the present invention couple
This is not construed as limiting.
Anti-human CTGF monoclonal's antibody after purification of acquisition is subjected to reduction SDS-PAGE electrophoresis detection, as a result such as Fig. 3
Shown, as seen from Figure 3, reducing agent, which opens the disulfide bond of antibody, is broken the heavy chain of antibody and light chain, forms two clear items
Band, and without other bands, illustrate anti-human CTGF monoclonal's antibody purity after purification it is high, without other foreign proteins.
The experiment of anti-human CTGF monoclonal's antibody of the embodiment of the present invention is described below in detail.
One, anti-human CTGF monoclonal's antibody titer detects.
The anti-human CTGF monoclonal's antibody after purification obtained in the present embodiment is subjected to potency using ELISA experimental method
Detection, it is known that anti-human CTGF monoclonal's antibody concentration is 100 μ g/mL.Method is as follows:
1, the ELISA Plate of appropriate hole count is taken to carry out antigen embedding, by each of suitable CTGF albumen coating to ELISA Plate
Hole；
2, it is cleaned ELISA Plate 3~5 times with rinsing liquid, it is anti-that the anti-human CTGF monoclonal after purification that the present embodiment obtains is added
Body, 37 DEG C of incubation 1h；
3, rinsing liquid cleans 5 times, be added with horseradish peroxidase mark or alkaline phosphatase target secondary antibody (for example,
Sheep anti-mouse igg), 37 DEG C of incubation 1h, rinsing liquid cleans 5 times；
4, be added substrate develop the color 10~20min, be added terminate liquid (for example, 2M sulfuric acid), terminate reaction, using microplate reader into
Row OD value is read, and the results are shown in Table 2.
Table 2: anti-human CTGF monoclonal's antibody titer testing result
Note: antibody 1 and antibody 2 are the anti-human CTGF monoclonal's antibody obtained in different mouse ascites.
As can be seen from Table 2, antibody 1 and 2 potency of antibody are all up 1:64000, and the potency of antibody 2 is better than antibody 1.
Two, the combination activity of protein immunoblot experiment detection anti-human CTGF monoclonal's antibody and CTGF albumen.
1, CTGF albumen is prepared, SDS-PAGE electrophoresis is carried out；
2, transferring film, NC film or pvdf membrane；
3, closing 2h is carried out with BSA or skimmed milk power；
4, it is added primary antibody (anti-human CTGF monoclonal's antibody 1 of preparation of the embodiment of the present invention), 37 DEG C, 1h；
5, it is cleaned 3~5 times, each 10min with rinsing liquid, addition ELIAS secondary antibody, 37 DEG C, 40min；
6, substrate reactions colour developing or ECL developing solution tabletting development is added.
As a result as shown in figure 4, from fig. 4, it can be seen that antibody 1 can specifically bind people's CTGF albumen, for anti-human CTGF antibody.
Three, anti-human CTGF monoclonal's antibody neutralization detection (Transwell method).
1, the low culture medium of serum content is put into the cell of upper layer.
2, the high culture medium of serum content is put into lower layer.It is respectively put into anti-human CTGF monoclonal's antibody 1, anti-human in a lower layer
CTGF monoclonal antibody 2 or IgG, blank are not put into antibody.
3, the film of bottom can be such that cell passes through in the cell of upper layer.
4, the cell invasion condition of production is observed after spreading rat smooth muscle cell, is photographed to record.
As a result as shown in figure 5, A is the culture of 10%FBS normal cell, lower layer is not put into antibody, by photo visible cell number
For the 1605/visual field；B is that (10%FBS+300 μ g/L lgG monoclonal is anti-for addition lgG monoclonal antibody in lower layer's culture medium
Body), it is the 998/visual field by photo visible cell number；C is that anti-human CTGF Dan Ke prepared by the present invention is added in lower layer's culture medium
Grand antibody 1 (10%FBS+300 μ g/L monoclonal antibody 1) is the 727/visual field by photo visible cell number；D is lower layer's culture
Anti-human CTGF monoclonal's antibody 2 (10%FBS+300 μ g/L monoclonal antibody 2) prepared by the present invention is added in base, it can by photo
See that cell number is the 562/visual field.
By above-mentioned experimental result, show that anti-human CTGF monoclonal's antibody of preparation of the embodiment of the present invention has high activity,
The invasive growth of rat smooth muscle cell can effectively be inhibited.
Anti-human CTGF monoclonal's antibody of preparation of the embodiment of the present invention has high activity, and CTGF can be inhibited to promote cell micro-
Pipe is formed and cell fibrosis, so that fibrosis be avoided to form caused vitreoretinopathy.
Although being particularly shown and describing the present invention, those skilled in the art referring to its exemplary embodiment
It should be understood that in the case where not departing from the spirit and scope of the present invention defined by claim form can be carried out to it
With the various changes in details.
<110>Liaoning He Shi medical college
<120>a kind of with inhibiting monoclonal antibody and preparation method thereof of vitreous-body-retina fibrosis lesion effect and answer
With
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2339
<212> DNA
<213>people CTGF(Connective tissue growth factor)
<400> 1
CACACAACAA CTCTTCCCCG CTGAGAGGAG ACAGCCAGTG CGACTCCACC CTCCAGCTCG 60
ACGGCAGCCG CCCCGGCCGA CAGCCCCGAG ACGACAGCCC GGCGCGTCCC GGTCCCCACC 120
TCCGACCACC GCCAGCGCTC CAGGCCCCGC CGCTCCCCGC TCGCCGCCAC CGCGCCCTCC 180
GCTCCGCCCG CAGTGCCAAC CATGACCGCC GCCAGTATGG GCCCCGTCCG CGTCGCCTTC 240
GTGGTCCTCC TCGCCCTCTG CAGCCGGCCG GCCGTCGGCC AGAACTGCAG CGGGCCGTGC 300
CGGTGCCCGG ACGAGCCGGC GCCGCGCTGC CCGGCGGGCG TGAGCCTCGT GCTGGACGGC 360
TGCGGCTGCT GCCGCGTCTG CGCCAAGCAG CTGGGCGAGC TGTGCACCGA GCGCGACCCA 420
TGCGACCCGC ACAAGGGCCT ATTCTGTCAC TTCGGCTCCC CGGCCAACCG CAAGATCGGC 480
GTGTGCACCG CCAAAGATGG TGCTCCCTGC ATCTTCGGTG GTACGGTGTA CCGCAGCGGA 540
GAGTCCTTCC AGAGCAGCTG CAAGTACCAG TGCACGTGCC TGGACGGGGC GGTGGGCTGC 600
ATGCCCCTGT GCAGCATGGA CGTTCGTCTG CCCAGCCCTG ACTGCCCCTT CCCGAGGAGG 660
GTCAAGCTGC CCGGGAAATG CTGCGAGGAG TGGGTGTGTG ACGAGCCCAA GGACCAAACC 720
GTGGTTGGGC CTGCCCTCGC GGCTTACCGA CTGGAAGACA CGTTTGGCCC AGACCCAACT 780
ATGATTAGAG CCAACTGCCT GGTCCAGACC ACAGAGTGGA GCGCCTGTTC CAAGACCTGT 840
GGGATGGGCA TCTCCACCCG GGTTACCAAT GACAACGCCT CCTGCAGGCT AGAGAAGCAG 900
AGCCGCCTGT GCATGGTCAG GCCTTGCGAA GCTGACCTGG AAGAGAACAT TAAGAAGGGC 960
AAAAAGTGCA TCCGTACTCC CAAAATCTCC AAGCCTATCA AGTTTGAGCT TTCTGGCTGC 1020
ACCAGCATGA AGACATACCG AGCTAAATTC TGTGGAGTAT GTACCGACGG CCGATGCTGC 1080
ACCCCCCACA GAACCACCAC CCTGCCGGTG GAGTTCAAGT GCCCTGACGG CGAGGTCATG 1140
AAGAAGAACA TGATGTTCAT CAAGACCTGT GCCTGCCATT ACAACTGTCC CGGAGACAAT 1200
GACATCTTTG AATCGCTGTA CTACAGGAAG ATGTACGGAG ACATGGCATG AAGCCAGAGA 1260
GTGAGAGACA TTAACTCATT AGACTGGAAC TTGAACTGAT TCACATCTCA TTTTTCCGTA 1320
AAAATGATTT CAGTAGCACA AGTTATTTAA ATCTGTTTTT CTAACTGGGG GAAAAGATTC 1380
CCACCCAATT CAAAACATTG TGCCATGTCA AACAAATAGT CTATCAACCC CAGACACTGG 1440
TTTGAAGAAT GTTAAGACTT GACAGTGGAA CTACATTAGT ACACAGCACC AGAATGTATA 1500
TTAAGGTGTG GCTTTAGGAG CAGTGGGAGG GTACCAGCAG AAAGGTTAGT ATCATCAGAT 1560
AGCATCTTAT ACGAGTAATA TGCCTGCTAT TTGAAGTGTA ATTGAGAAGG AAAATTTTAG 1620
CGTGCTCACT GACCTGCCTG TAGCCCCAGT GACAGCTAGG ATGTGCATTC TCCAGCCATC 1680
AAGAGACTGA GTCAAGTTGT TCCTTAAGTC AGAACAGCAG ACTCAGCTCT GACATTCTGA 1740
TTCGAATGAC ACTGTTCAGG AATCGGAATC CTGTCGATTA GACTGGACAG CTTGTGGCAA 1800
GTGAATTTGC CTGTAACAAG CCAGATTTTT TAAAATTTAT ATTGTAAATA TTGTGTGTGT 1860
GTGTGTGTGT GTATATATAT ATATATGTAC AGTTATCTAA GTTAATTTAA AGTTGTTTGT 1920
GCCTTTTTAT TTTTGTTTTT AATGCTTTGA TATTTCAATG TTAGCCTCAA TTTCTGAACA 1980
CCATAGGTAG AATGTAAAGC TTGTCTGATC GTTCAAAGCA TGAAATGGAT ACTTATATGG 2040
AAATTCTGCT CAGATAGAAT GACAGTCCGT CAAAACAGAT TGTTTGCAAA GGGGAGGCAT 2100
CAGTGTCCTT GGCAGGCTGA TTTCTAGGTA GGAAATGTGG TAGCCTCACT TTTAATGAAC 2160
AAATGGCCTT TATTAAAAAC TGAGTGACTC TATATAGCTG ATCAGTTTTT TCACCTGGAA 2220
GCATTTGTTT CTACTTTGAT ATGACTGTTT TTCGGACAGT TTATTTGTTG AGAGTGTGAC 2280
CAAAAGTTAC ATGTTTGCAC CTTTCTAGTT GAAAATAAAG TGTATATTTT TTCTATAAA 2339
<210> 2
<211> 349
<212> PRT
<213>people CTGF(Connective tissue growth factor)
<400> 2
Met Thr Ala Ala Ser Met Gly Pro Val Arg Val Ala Phe Val Val Leu Leu Ala
1 5 10 15
Leu Cys Ser Arg Pro Ala Val Gly Gln Asn Cys Ser Gly Pro Cys Arg Cys Pro
20 25 30 35
Asp Glu Pro Ala Pro Arg Cys Pro Ala Gly Val Ser Leu Val Leu Asp Gly Cys
40 45 50
Gly Cys Cys Arg Val Cys Ala Lys Gln Leu Gly Glu Leu Cys Thr Glu Arg Asp
55 60 65 70
Pro Cys Asp Pro His Lys Gly Leu Phe Cys His Phe Gly Ser Pro Ala Asn Arg
75 80 85 90
Lys Ile Gly Val Cys Thr Ala Lys Asp Gly Ala Pro Cys Ile Phe Gly Gly Thr
95 100 105
Val Tyr Arg Ser Gly Glu Ser Phe Gln Ser Ser Cys Lys Tyr Gln Cys Thr Cys
110 115 120 125
Leu Asp Gly Ala Val Gly Cys Met Pro Leu Cys Ser Met Asp Val Arg Leu Pro
130 135 140
Ser Pro Asp Cys Pro Phe Pro Arg Arg Val Lys Leu Pro Gly Lys Cys Cys Glu
145 150 155 160
Glu Trp Val Cys Asp Glu Pro Lys Asp Gln Thr Val Val Gly Pro Ala Leu Ala
165 170 175 180
Ala Tyr Arg Leu Glu Asp Thr Phe Gly Pro Asp Pro Thr Met Ile Arg Ala Asn
185 190 195
Cys Leu Val Gln Thr Thr Glu Trp Ser Ala Cys Ser Lys Thr Cys Gly Met Gly
200 205 210 215
Ile Ser Thr Arg Val Thr Asn Asp Asn Ala Ser Cys Arg Leu Glu Lys Gln Ser
220 225 230
Arg Leu Cys Met Val Arg Pro Cys Glu Ala Asp Leu Glu Glu Asn Ile Lys Lys
235 240 245 250
Gly Lys Lys Cys Ile Arg Thr Pro Lys Ile Ser Lys Pro Ile Lys Phe Glu Leu
255 260 265 270
Ser Gly Cys Thr Ser Met Lys Thr Tyr Arg Ala Lys Phe Cys Gly Val Cys Thr
275 280 285
Asp Gly Arg Cys Cys Thr Pro His Arg Thr Thr Thr Leu Pro Val Glu Phe Lys
290 295 300 305
Cys Pro Asp Gly Glu Val Met Lys Lys Asn Met Met Phe Ile Lys Thr Cys Ala
310 315 320
Cys His Tyr Asn Cys Pro Gly Asp Asn Asp Ile Phe Glu Ser Leu Tyr Tyr Arg
325 330 335 340
Lys Met Tyr Gly Asp Met Ala
345
Claims (8)
1. a kind of with the monoclonal antibody for inhibiting the effect of vitreous-body-retina fibrosis lesion, which is characterized in that the Dan Ke
Grand antibody is anti-human CTGF monoclonal's antibody, and the antibody fragment of anti-human CTGF monoclonal's antibody includes single-stranded variable region piece
Section, Fab, Fab ', F (ab ') 2 or nano antibody.
2. monoclonal antibody according to claim 1, which is characterized in that the antibody fragment and other peptide or protein matter
Merge or be modified agent modification.
3. a kind of preparation method with the monoclonal antibody for inhibiting the effect of vitreous-body-retina fibrosis lesion, feature exist
In, comprising:
Obtain nucleic acid sequence recombined human CTGF antigen protein as shown in SEQ ID NO:1；
Mouse is immunized with the recombined human CTGF antigen protein, and separation obtains mouse boosting cell in the mouse after being immunized；
The mouse boosting cell is mixed with homology myeloma cell, film fusion is carried out using PEG, it is thin to form hybridoma
Born of the same parents；
The hybridoma for screening successfully film fusion, carries out in-vitro multiplication culture, hybridoma is inoculated in mouse peritoneal, in advance
The monoclonal antibody is obtained after fixing time from the ascites of the mouse after inoculation.
4. preparation method according to claim 3, which is characterized in that obtain the list from the ascites of the mouse after inoculation
The step of clonal antibody includes:
The ascites is slightly purified；
It dialyses to the product slightly purified；
It is purified using product of the HPLC method to the dialysis,
Wherein, the purifying uses anionic exchange medium, and using mobile phase A and Mobile phase B as eluent, flow velocity is 1~3mL/
Min is eluted using gradient elution mode, and 35~40 DEG C of column temperature, Detection wavelength 280nm；
The mobile phase A be TRIS buffer, the Mobile phase B be TRIS buffer and
The mixed solution of sodium chloride.
5. preparation method according to claim 3 or 4, which is characterized in that immune with the recombined human CTGF antigen protein
The step of animal includes:
Freund's complete adjuvant is added in the recombined human CTGF antigen protein, first immunisation injection is carried out to mouse；
Incomplete Freund's adjuvant is added in the recombined human CTGF antigen protein, the mouse after first immunisation is carried out reinforcing exempting from
Epidemic disease injection.
6. preparation method according to claim 3 or 4, which is characterized in that press 1 × 108A mouse boosting cell and 2~3
×107A homology myeloma cell mixes.
7. application of the monoclonal antibody described in claim 1 in preparation treatment vitreous-body-retina fibrosis lesion drug.
8. application according to claim 7, which is characterized in that the vitreous-body-retina fibrosis lesion includes: green light
Caused vitreous-body-retina after eye filtering surgery, eye traumas, diabetic retinopathy and vitrectomy
Fibrosis lesion.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910197628.8A CN109824777A (en) | 2019-03-15 | 2019-03-15 | A kind of monoclonal antibody and its preparation method and application with inhibition vitreous-body-retina fibrosis lesion effect |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910197628.8A CN109824777A (en) | 2019-03-15 | 2019-03-15 | A kind of monoclonal antibody and its preparation method and application with inhibition vitreous-body-retina fibrosis lesion effect |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109824777A true CN109824777A (en) | 2019-05-31 |
Family
ID=66870805
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910197628.8A Pending CN109824777A (en) | 2019-03-15 | 2019-03-15 | A kind of monoclonal antibody and its preparation method and application with inhibition vitreous-body-retina fibrosis lesion effect |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109824777A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112626063A (en) * | 2020-12-18 | 2021-04-09 | 上海药明生物技术有限公司 | Method for preparing hybridoma by enriching mouse plasma cells by using CD138+ biomarker and application |
| WO2024079310A1 (en) * | 2022-10-14 | 2024-04-18 | Ebbil, Ltd. | Sil-6r and ctgf binding proteins and methods of use thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1829740A (en) * | 2003-06-04 | 2006-09-06 | 菲布罗根公司 | Connective tissue growth factor antibodies |
| CN104011206A (en) * | 2011-12-22 | 2014-08-27 | 安斯泰来制药株式会社 | Novel anti-human ctgf antibody |
-
2019
- 2019-03-15 CN CN201910197628.8A patent/CN109824777A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1829740A (en) * | 2003-06-04 | 2006-09-06 | 菲布罗根公司 | Connective tissue growth factor antibodies |
| CN104011206A (en) * | 2011-12-22 | 2014-08-27 | 安斯泰来制药株式会社 | Novel anti-human ctgf antibody |
Non-Patent Citations (2)
| Title |
|---|
| JEFFREY R.DESCHAMPS等: "A high-performance liquid chromatographic procedure for the purification of mouse monoclonal antibodies", 《ANALYTICAL BIOCHEMISTRY》 * |
| NATIONAL CENTER FOR BIOTECHNOLOGY INFORMATION: "NCBI Reference Sequence: NP_001901.3", 《NATIONAL CENTER FOR BIOTECHNOLOGY INFORMATION》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112626063A (en) * | 2020-12-18 | 2021-04-09 | 上海药明生物技术有限公司 | Method for preparing hybridoma by enriching mouse plasma cells by using CD138+ biomarker and application |
| WO2024079310A1 (en) * | 2022-10-14 | 2024-04-18 | Ebbil, Ltd. | Sil-6r and ctgf binding proteins and methods of use thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| KR102179674B1 (en) | Anti-EGFR and anti-CD3 bispecific antibodies and their applications | |
| EP0413908B2 (en) | Soluble extracellular fragment of the human IFN-beta 2/IL-6 receptor, its preparation and pharmaceutical compositions containing it | |
| CA2346997C (en) | Cytotoxic lymphocyte maturation factor and monoclonal antibodies directed thereto | |
| ES2206448T3 (en) | HEREGULINS (HRGS), PROTEINS OF UNION OF P185? ERB2. | |
| EP3766901A1 (en) | Antibody targeting ctla-4 , preparation method therefor and use thereof | |
| JP6006404B2 (en) | Anti-BLyS antibody | |
| CN112638406A (en) | Interleukin-2 variants and methods of use thereof | |
| JPH06502997A (en) | Nucleotide sequence encoding the variable region of the β chain of human T lymphocyte receptor and corresponding peptide segment and use in diagnosis and therapy | |
| EA003086B1 (en) | Polypeptides lag-3v1, lag-3v2 and lag-3v3 and encoding them to dna | |
| AU2006310882A1 (en) | Neurotrophin-derived peptide sequences | |
| CA3077304C (en) | Anti-pacap antibody | |
| CN109071673A (en) | Anti- PCSK9 antibody and its application | |
| RU2716101C2 (en) | Sclerostin antibody, its antigen-binding fragment and medical application | |
| CN109879963A (en) | A kind of monoclonal antibody and its preparation method and application for inhibiting histoorgan fibrosis and new vessels to be formed | |
| CN109824777A (en) | A kind of monoclonal antibody and its preparation method and application with inhibition vitreous-body-retina fibrosis lesion effect | |
| CN114591434B (en) | anti-Siglec 15 antibody and preparation method and application thereof | |
| CN100586960C (en) | HAb18GC2 monoclonal antibody and its light and heavy chain variable area genes, and application | |
| CN114369161B (en) | MICA antibodies and uses thereof | |
| CN105916883A (en) | Bifunctional fusion protein, preparation method therefor, and use thereof | |
| CN101864398B (en) | Monoclonal antibody of anti-gonadotropin-releasing hormone receptor and application thereof | |
| WO2019192493A1 (en) | Anti-human lag-3 monoclonal antibody and use thereof | |
| WO2023046097A1 (en) | Anti-tigit humanized antibody or antigen-binding fragment thereof and application thereof | |
| KR20210032412A (en) | Peripheral nerve disorder, peripheral nerve disorder, or a method of preventing or treating pain due to a disease recognized as a stellate cell disorder | |
| US20060275865A1 (en) | Autotaxin: motility stimulating protein useful in cancer diagnosis and therapy | |
| KR102090160B1 (en) | Specific antigen purification method and monoclonal antibody production method using the same |
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 | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190531 |
|
| RJ01 | Rejection of invention patent application after publication |