CN104311654A - CIDE3 polypeptide, preparation of antibody thereof and applications of the antibody - Google Patents
CIDE3 polypeptide, preparation of antibody thereof and applications of the antibody Download PDFInfo
- Publication number
- CN104311654A CN104311654A CN201410391896.0A CN201410391896A CN104311654A CN 104311654 A CN104311654 A CN 104311654A CN 201410391896 A CN201410391896 A CN 201410391896A CN 104311654 A CN104311654 A CN 104311654A
- Authority
- CN
- China
- Prior art keywords
- cide3
- antibody
- protein
- polypeptide
- polyclonal antibody
- 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
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 43
- 229920001184 polypeptide Polymers 0.000 title abstract description 23
- 102000004196 processed proteins & peptides Human genes 0.000 title abstract description 23
- 238000002360 preparation method Methods 0.000 title abstract description 3
- 102000036639 antigens Human genes 0.000 claims abstract description 28
- 108091007433 antigens Proteins 0.000 claims abstract description 28
- 239000000427 antigen Substances 0.000 claims abstract description 19
- 208000014018 liver neoplasm Diseases 0.000 claims abstract description 13
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 2
- 241000283973 Oryctolagus cuniculus Species 0.000 claims description 18
- 230000008878 coupling Effects 0.000 claims description 18
- 238000010168 coupling process Methods 0.000 claims description 18
- 238000005859 coupling reaction Methods 0.000 claims description 18
- 108010078791 Carrier Proteins Proteins 0.000 claims description 13
- 102000014914 Carrier Proteins Human genes 0.000 claims description 13
- 108010045069 keyhole-limpet hemocyanin Proteins 0.000 claims description 7
- 206010019695 Hepatic neoplasm Diseases 0.000 claims description 6
- 239000003550 marker Substances 0.000 claims description 6
- 101000800130 Bos taurus Thyroglobulin Proteins 0.000 claims description 5
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 5
- 108010058846 Ovalbumin Proteins 0.000 claims description 5
- 229940098773 bovine serum albumin Drugs 0.000 claims description 5
- 229940092253 ovalbumin Drugs 0.000 claims description 5
- 108090000623 proteins and genes Proteins 0.000 abstract description 26
- 102000004169 proteins and genes Human genes 0.000 abstract description 19
- 201000007270 liver cancer Diseases 0.000 abstract description 7
- 101000775558 Homo sapiens Cell death activator CIDE-3 Proteins 0.000 abstract description 4
- 238000004458 analytical method Methods 0.000 abstract description 4
- 238000001514 detection method Methods 0.000 abstract description 4
- 238000004393 prognosis Methods 0.000 abstract description 4
- 238000003745 diagnosis Methods 0.000 abstract description 2
- 102000053843 human CIDEC Human genes 0.000 abstract 2
- 230000000694 effects Effects 0.000 abstract 1
- 238000010353 genetic engineering Methods 0.000 abstract 1
- 230000035945 sensitivity Effects 0.000 abstract 1
- 230000001225 therapeutic effect Effects 0.000 abstract 1
- 235000018102 proteins Nutrition 0.000 description 15
- 150000001413 amino acids Chemical class 0.000 description 12
- 239000007788 liquid Substances 0.000 description 12
- 238000000034 method Methods 0.000 description 11
- 239000002953 phosphate buffered saline Substances 0.000 description 10
- 238000012797 qualification Methods 0.000 description 10
- 239000000872 buffer Substances 0.000 description 9
- 235000001014 amino acid Nutrition 0.000 description 8
- 206010028980 Neoplasm Diseases 0.000 description 7
- 230000015572 biosynthetic process Effects 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- 238000003786 synthesis reaction Methods 0.000 description 7
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 6
- 238000002965 ELISA Methods 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 210000004899 c-terminal region Anatomy 0.000 description 6
- 210000004027 cell Anatomy 0.000 description 6
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 6
- 210000002966 serum Anatomy 0.000 description 6
- 238000010790 dilution Methods 0.000 description 5
- 239000012895 dilution Substances 0.000 description 5
- 239000012153 distilled water Substances 0.000 description 5
- 238000000746 purification Methods 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 4
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 4
- 230000006907 apoptotic process Effects 0.000 description 4
- 238000011033 desalting Methods 0.000 description 4
- 238000001962 electrophoresis Methods 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 239000013642 negative control Substances 0.000 description 4
- 238000004321 preservation Methods 0.000 description 4
- 239000000523 sample Substances 0.000 description 4
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 4
- 102100032137 Cell death activator CIDE-3 Human genes 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 229920002684 Sepharose Polymers 0.000 description 3
- YUJLIIRMIAGMCQ-CIUDSAMLSA-N Ser-Leu-Ser Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O YUJLIIRMIAGMCQ-CIUDSAMLSA-N 0.000 description 3
- 230000004913 activation Effects 0.000 description 3
- 239000002671 adjuvant Substances 0.000 description 3
- 238000001261 affinity purification Methods 0.000 description 3
- 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 3
- 201000011510 cancer Diseases 0.000 description 3
- 230000030833 cell death Effects 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000013467 fragmentation Methods 0.000 description 3
- 238000006062 fragmentation reaction Methods 0.000 description 3
- 108010055341 glutamyl-glutamic acid Proteins 0.000 description 3
- 230000036039 immunity Effects 0.000 description 3
- 210000005229 liver cell Anatomy 0.000 description 3
- 238000011068 loading method Methods 0.000 description 3
- 239000006166 lysate Substances 0.000 description 3
- 238000001556 precipitation Methods 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 239000011734 sodium Substances 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 238000001262 western blot Methods 0.000 description 3
- 238000009736 wetting Methods 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 239000004471 Glycine Substances 0.000 description 2
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- PEEHTFAAVSWFBL-UHFFFAOYSA-N Maleimide Chemical compound O=C1NC(=O)C=C1 PEEHTFAAVSWFBL-UHFFFAOYSA-N 0.000 description 2
- 241000283977 Oryctolagus Species 0.000 description 2
- 229920001213 Polysorbate 20 Polymers 0.000 description 2
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 2
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
- 239000011543 agarose gel Substances 0.000 description 2
- 238000011091 antibody purification Methods 0.000 description 2
- 230000001640 apoptogenic effect Effects 0.000 description 2
- 230000008033 biological extinction Effects 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 238000010241 blood sampling Methods 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 235000019846 buffering salt Nutrition 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000013016 damping Methods 0.000 description 2
- 229940088598 enzyme Drugs 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 235000011187 glycerol Nutrition 0.000 description 2
- 210000005260 human cell Anatomy 0.000 description 2
- 230000002209 hydrophobic effect Effects 0.000 description 2
- 230000005847 immunogenicity Effects 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 2
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 2
- 239000001632 sodium acetate Substances 0.000 description 2
- 229960004249 sodium acetate Drugs 0.000 description 2
- 235000017281 sodium acetate Nutrition 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- YRNWIFYIFSBPAU-UHFFFAOYSA-N 4-[4-(dimethylamino)phenyl]-n,n-dimethylaniline Chemical compound C1=CC(N(C)C)=CC=C1C1=CC=C(N(C)C)C=C1 YRNWIFYIFSBPAU-UHFFFAOYSA-N 0.000 description 1
- UCIYCBSJBQGDGM-LPEHRKFASA-N Ala-Arg-Pro Chemical compound C[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N1CCC[C@@H]1C(=O)O)N UCIYCBSJBQGDGM-LPEHRKFASA-N 0.000 description 1
- PJNSIUPOXFBHDM-GUBZILKMSA-N Ala-Arg-Val Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C(C)C)C(O)=O PJNSIUPOXFBHDM-GUBZILKMSA-N 0.000 description 1
- LDLSENBXQNDTPB-DCAQKATOSA-N Ala-Lys-Arg Chemical compound NCCCC[C@H](NC(=O)[C@@H](N)C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N LDLSENBXQNDTPB-DCAQKATOSA-N 0.000 description 1
- VCSABYLVNWQYQE-SRVKXCTJSA-N Ala-Lys-Lys Chemical compound NCCCC[C@H](NC(=O)[C@@H](N)C)C(=O)N[C@@H](CCCCN)C(O)=O VCSABYLVNWQYQE-SRVKXCTJSA-N 0.000 description 1
- VCSABYLVNWQYQE-UHFFFAOYSA-N Ala-Lys-Lys Natural products NCCCCC(NC(=O)C(N)C)C(=O)NC(CCCCN)C(O)=O VCSABYLVNWQYQE-UHFFFAOYSA-N 0.000 description 1
- GKAZXNDATBWNBI-DCAQKATOSA-N Ala-Met-Lys Chemical compound C[C@@H](C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCCN)C(=O)O)N GKAZXNDATBWNBI-DCAQKATOSA-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
- NCQMBSJGJMYKCK-ZLUOBGJFSA-N Ala-Ser-Ser Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O NCQMBSJGJMYKCK-ZLUOBGJFSA-N 0.000 description 1
- LSMDIAAALJJLRO-XQXXSGGOSA-N Ala-Thr-Glu Chemical compound [H]N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(O)=O)C(O)=O LSMDIAAALJJLRO-XQXXSGGOSA-N 0.000 description 1
- UPKMBGAAEZGHOC-RWMBFGLXSA-N Arg-His-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC2=CN=CN2)NC(=O)[C@H](CCCN=C(N)N)N)C(=O)O UPKMBGAAEZGHOC-RWMBFGLXSA-N 0.000 description 1
- NGTYEHIRESTSRX-UWVGGRQHSA-N Arg-Lys-Gly Chemical compound NCCCC[C@@H](C(=O)NCC(O)=O)NC(=O)[C@@H](N)CCCN=C(N)N NGTYEHIRESTSRX-UWVGGRQHSA-N 0.000 description 1
- OQPAZKMGCWPERI-GUBZILKMSA-N Arg-Ser-Val Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(O)=O OQPAZKMGCWPERI-GUBZILKMSA-N 0.000 description 1
- INOIAEUXVVNJKA-XGEHTFHBSA-N Arg-Thr-Ser Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(O)=O INOIAEUXVVNJKA-XGEHTFHBSA-N 0.000 description 1
- DRDWXKWUSIKKOB-PJODQICGSA-N Arg-Trp-Ala Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](C)C(O)=O DRDWXKWUSIKKOB-PJODQICGSA-N 0.000 description 1
- XMHFCUKJRCQXGI-CIUDSAMLSA-N Asn-Pro-Gln Chemical compound C1C[C@H](N(C1)C(=O)[C@H](CC(=O)N)N)C(=O)N[C@@H](CCC(=O)N)C(=O)O XMHFCUKJRCQXGI-CIUDSAMLSA-N 0.000 description 1
- PCJOFZYFFMBZKC-PCBIJLKTSA-N Asp-Phe-Ile Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O PCJOFZYFFMBZKC-PCBIJLKTSA-N 0.000 description 1
- JDDYEZGPYBBPBN-JRQIVUDYSA-N Asp-Thr-Tyr Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O JDDYEZGPYBBPBN-JRQIVUDYSA-N 0.000 description 1
- ITGFVUYOLWBPQW-KKHAAJSZSA-N Asp-Thr-Val 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)C)C(O)=O ITGFVUYOLWBPQW-KKHAAJSZSA-N 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
- 101710123595 Cell death activator CIDE-3 Proteins 0.000 description 1
- 102100032141 Cell death activator CIDE-A Human genes 0.000 description 1
- YFXFOZPXVFPBDH-VZFHVOOUSA-N Cys-Ala-Thr Chemical compound C[C@@H](O)[C@H](NC(=O)[C@H](C)NC(=O)[C@@H](N)CS)C(O)=O YFXFOZPXVFPBDH-VZFHVOOUSA-N 0.000 description 1
- XGIAHEUULGOZHH-GUBZILKMSA-N Cys-Arg-Val Chemical compound CC(C)[C@@H](C(=O)O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CS)N XGIAHEUULGOZHH-GUBZILKMSA-N 0.000 description 1
- ZJBWJHQDOIMVLM-WHFBIAKZSA-N Cys-Cys-Gly Chemical compound SC[C@H](N)C(=O)N[C@@H](CS)C(=O)NCC(O)=O ZJBWJHQDOIMVLM-WHFBIAKZSA-N 0.000 description 1
- 239000003109 Disodium ethylene diamine tetraacetate Substances 0.000 description 1
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 description 1
- XXLBHPPXDUWYAG-XQXXSGGOSA-N Gln-Ala-Thr Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O XXLBHPPXDUWYAG-XQXXSGGOSA-N 0.000 description 1
- KVXVVDFOZNYYKZ-DCAQKATOSA-N Gln-Gln-Leu Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(O)=O KVXVVDFOZNYYKZ-DCAQKATOSA-N 0.000 description 1
- JXFLPKSDLDEOQK-JHEQGTHGSA-N Gln-Gly-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CCC(N)=O JXFLPKSDLDEOQK-JHEQGTHGSA-N 0.000 description 1
- WLRYGVYQFXRJDA-DCAQKATOSA-N Gln-Pro-Pro Chemical compound NC(=O)CC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N1[C@H](C(O)=O)CCC1 WLRYGVYQFXRJDA-DCAQKATOSA-N 0.000 description 1
- ATRHMOJQJWPVBQ-DRZSPHRISA-N Glu-Ala-Phe Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O ATRHMOJQJWPVBQ-DRZSPHRISA-N 0.000 description 1
- DSPQRJXOIXHOHK-WDSKDSINSA-N Glu-Asp-Gly Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(O)=O DSPQRJXOIXHOHK-WDSKDSINSA-N 0.000 description 1
- SJPMNHCEWPTRBR-BQBZGAKWSA-N Glu-Glu-Gly Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(O)=O SJPMNHCEWPTRBR-BQBZGAKWSA-N 0.000 description 1
- WRNAXCVRSBBKGS-BQBZGAKWSA-N Glu-Gly-Gln Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H](CCC(N)=O)C(O)=O WRNAXCVRSBBKGS-BQBZGAKWSA-N 0.000 description 1
- QCMVGXDELYMZET-GLLZPBPUSA-N Glu-Thr-Glu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(O)=O)C(O)=O QCMVGXDELYMZET-GLLZPBPUSA-N 0.000 description 1
- IXKRSKPKSLXIHN-YUMQZZPRSA-N Gly-Cys-Leu Chemical compound [H]NCC(=O)N[C@@H](CS)C(=O)N[C@@H](CC(C)C)C(O)=O IXKRSKPKSLXIHN-YUMQZZPRSA-N 0.000 description 1
- AQLHORCVPGXDJW-IUCAKERBSA-N Gly-Gln-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)CN AQLHORCVPGXDJW-IUCAKERBSA-N 0.000 description 1
- ALOBJFDJTMQQPW-ONGXEEELSA-N Gly-His-Val Chemical compound CC(C)[C@@H](C(=O)O)NC(=O)[C@H](CC1=CN=CN1)NC(=O)CN ALOBJFDJTMQQPW-ONGXEEELSA-N 0.000 description 1
- RVKIPWVMZANZLI-UHFFFAOYSA-N H-Lys-Trp-OH Natural products C1=CC=C2C(CC(NC(=O)C(N)CCCCN)C(O)=O)=CNC2=C1 RVKIPWVMZANZLI-UHFFFAOYSA-N 0.000 description 1
- TTYKEFZRLKQTHH-MELADBBJSA-N His-Lys-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCCCN)NC(=O)[C@H](CC2=CN=CN2)N)C(=O)O TTYKEFZRLKQTHH-MELADBBJSA-N 0.000 description 1
- 101000775570 Homo sapiens Cell death activator CIDE-A Proteins 0.000 description 1
- LLZLRXBTOOFODM-QSFUFRPTSA-N Ile-Asp-Val Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](C(C)C)C(=O)O)N LLZLRXBTOOFODM-QSFUFRPTSA-N 0.000 description 1
- MASWXTFJVNRZPT-NAKRPEOUSA-N Ile-Met-Ala Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCSC)C(=O)N[C@@H](C)C(=O)O)N MASWXTFJVNRZPT-NAKRPEOUSA-N 0.000 description 1
- ZUPJCJINYQISSN-XUXIUFHCSA-N Ile-Met-Lys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCCN)C(=O)O)N ZUPJCJINYQISSN-XUXIUFHCSA-N 0.000 description 1
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 1
- 241000880493 Leptailurus serval Species 0.000 description 1
- WNGVUZWBXZKQES-YUMQZZPRSA-N Leu-Ala-Gly Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C)C(=O)NCC(O)=O WNGVUZWBXZKQES-YUMQZZPRSA-N 0.000 description 1
- WGNOPSQMIQERPK-UHFFFAOYSA-N Leu-Asn-Pro Natural products CC(C)CC(N)C(=O)NC(CC(=O)N)C(=O)N1CCCC1C(=O)O WGNOPSQMIQERPK-UHFFFAOYSA-N 0.000 description 1
- KAFOIVJDVSZUMD-DCAQKATOSA-N Leu-Gln-Gln Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O KAFOIVJDVSZUMD-DCAQKATOSA-N 0.000 description 1
- KAFOIVJDVSZUMD-UHFFFAOYSA-N Leu-Gln-Gln Natural products CC(C)CC(N)C(=O)NC(CCC(N)=O)C(=O)NC(CCC(N)=O)C(O)=O KAFOIVJDVSZUMD-UHFFFAOYSA-N 0.000 description 1
- FQZPTCNSNPWHLJ-AVGNSLFASA-N Leu-Gln-Lys Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCCN)C(O)=O FQZPTCNSNPWHLJ-AVGNSLFASA-N 0.000 description 1
- PDQDCFBVYXEFSD-SRVKXCTJSA-N Leu-Leu-Asp Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O PDQDCFBVYXEFSD-SRVKXCTJSA-N 0.000 description 1
- YOKVEHGYYQEQOP-QWRGUYRKSA-N Leu-Leu-Gly Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)NCC(O)=O YOKVEHGYYQEQOP-QWRGUYRKSA-N 0.000 description 1
- FKQPWMZLIIATBA-AJNGGQMLSA-N Leu-Lys-Ile Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O FKQPWMZLIIATBA-AJNGGQMLSA-N 0.000 description 1
- AXVIGSRGTMNSJU-YESZJQIVSA-N Leu-Tyr-Pro Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N2CCC[C@@H]2C(=O)O)N AXVIGSRGTMNSJU-YESZJQIVSA-N 0.000 description 1
- UWKNTTJNVSYXPC-CIUDSAMLSA-N Lys-Ala-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CCCCN UWKNTTJNVSYXPC-CIUDSAMLSA-N 0.000 description 1
- NKKFVJRLCCUJNA-QWRGUYRKSA-N Lys-Gly-Lys Chemical compound NCCCC[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCCCN NKKFVJRLCCUJNA-QWRGUYRKSA-N 0.000 description 1
- IOQWIOPSKJOEKI-SRVKXCTJSA-N Lys-Ser-Leu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(O)=O IOQWIOPSKJOEKI-SRVKXCTJSA-N 0.000 description 1
- STTRPDDKDVKIDF-KKUMJFAQSA-N Met-Glu-Tyr Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 STTRPDDKDVKIDF-KKUMJFAQSA-N 0.000 description 1
- 108010079364 N-glycylalanine Proteins 0.000 description 1
- 108010087066 N2-tryptophyllysine Proteins 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 240000007711 Peperomia pellucida Species 0.000 description 1
- RIYZXJVARWJLKS-KKUMJFAQSA-N Phe-Asp-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](N)CC1=CC=CC=C1 RIYZXJVARWJLKS-KKUMJFAQSA-N 0.000 description 1
- OKQQWSNUSQURLI-JYJNAYRXSA-N Phe-Met-Val Chemical compound CC(C)[C@@H](C(=O)O)NC(=O)[C@H](CCSC)NC(=O)[C@H](CC1=CC=CC=C1)N OKQQWSNUSQURLI-JYJNAYRXSA-N 0.000 description 1
- AXIOGMQCDYVTNY-ACRUOGEOSA-N Phe-Phe-Leu Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(O)=O)NC(=O)[C@@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 AXIOGMQCDYVTNY-ACRUOGEOSA-N 0.000 description 1
- GKRCCTYAGQPMMP-IHRRRGAJSA-N Phe-Ser-Met Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCSC)C(O)=O GKRCCTYAGQPMMP-IHRRRGAJSA-N 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- PCWLNNZTBJTZRN-AVGNSLFASA-N Pro-Pro-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@H]1NCCC1 PCWLNNZTBJTZRN-AVGNSLFASA-N 0.000 description 1
- FNGOXVQBBCMFKV-CIUDSAMLSA-N Pro-Ser-Glu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(O)=O FNGOXVQBBCMFKV-CIUDSAMLSA-N 0.000 description 1
- MDAWMJUZHBQTBO-XGEHTFHBSA-N Pro-Thr-Cys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@@H]1CCCN1)O MDAWMJUZHBQTBO-XGEHTFHBSA-N 0.000 description 1
- 101710093543 Probable non-specific lipid-transfer protein Proteins 0.000 description 1
- VQBLHWSPVYYZTB-DCAQKATOSA-N Ser-Arg-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CO)N VQBLHWSPVYYZTB-DCAQKATOSA-N 0.000 description 1
- UFKPDBLKLOBMRH-XHNCKOQMSA-N Ser-Glu-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CO)N)C(=O)O UFKPDBLKLOBMRH-XHNCKOQMSA-N 0.000 description 1
- HEUVHBXOVZONPU-BJDJZHNGSA-N Ser-Leu-Ile Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O HEUVHBXOVZONPU-BJDJZHNGSA-N 0.000 description 1
- NMZXJDSKEGFDLJ-DCAQKATOSA-N Ser-Pro-Lys Chemical compound C1C[C@H](N(C1)C(=O)[C@H](CO)N)C(=O)N[C@@H](CCCCN)C(=O)O NMZXJDSKEGFDLJ-DCAQKATOSA-N 0.000 description 1
- 239000006180 TBST buffer Substances 0.000 description 1
- NJEMRSFGDNECGF-GCJQMDKQSA-N Thr-Ala-Asp Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CC(O)=O NJEMRSFGDNECGF-GCJQMDKQSA-N 0.000 description 1
- MEBDIIKMUUNBSB-RPTUDFQQSA-N Thr-Phe-Tyr Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O MEBDIIKMUUNBSB-RPTUDFQQSA-N 0.000 description 1
- NBIIPOKZPUGATB-BWBBJGPYSA-N Thr-Ser-Cys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CS)C(=O)O)N)O NBIIPOKZPUGATB-BWBBJGPYSA-N 0.000 description 1
- LECUEEHKUFYOOV-ZJDVBMNYSA-N Thr-Thr-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@@H](N)[C@@H](C)O LECUEEHKUFYOOV-ZJDVBMNYSA-N 0.000 description 1
- DXHHCIYKHRKBOC-BHYGNILZSA-N Trp-Gln-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CC2=CNC3=CC=CC=C32)N)C(=O)O DXHHCIYKHRKBOC-BHYGNILZSA-N 0.000 description 1
- RCLOWEZASFJFEX-KKUMJFAQSA-N Tyr-Asp-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 RCLOWEZASFJFEX-KKUMJFAQSA-N 0.000 description 1
- FMXFHNSFABRVFZ-BZSNNMDCSA-N Tyr-Lys-Leu Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(O)=O FMXFHNSFABRVFZ-BZSNNMDCSA-N 0.000 description 1
- FDKDGFGTHGJKNV-FHWLQOOXSA-N Tyr-Phe-Gln Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)O)NC(=O)[C@H](CC2=CC=C(C=C2)O)N FDKDGFGTHGJKNV-FHWLQOOXSA-N 0.000 description 1
- MQGGXGKQSVEQHR-KKUMJFAQSA-N Tyr-Ser-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 MQGGXGKQSVEQHR-KKUMJFAQSA-N 0.000 description 1
- LYERIXUFCYVFFX-GVXVVHGQSA-N Val-Leu-Glu Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)O)NC(=O)[C@H](C(C)C)N LYERIXUFCYVFFX-GVXVVHGQSA-N 0.000 description 1
- RWOGENDAOGMHLX-DCAQKATOSA-N Val-Lys-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C(C)C)N RWOGENDAOGMHLX-DCAQKATOSA-N 0.000 description 1
- NZYNRRGJJVSSTJ-GUBZILKMSA-N Val-Ser-Val Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(O)=O NZYNRRGJJVSSTJ-GUBZILKMSA-N 0.000 description 1
- JVGDAEKKZKKZFO-RCWTZXSCSA-N Val-Val-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](C(C)C)N)O JVGDAEKKZKKZFO-RCWTZXSCSA-N 0.000 description 1
- DPDMMXDBJGCCQC-UHFFFAOYSA-N [Na].[Cl] Chemical compound [Na].[Cl] DPDMMXDBJGCCQC-UHFFFAOYSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 238000003450 affinity purification method Methods 0.000 description 1
- 239000005557 antagonist Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 230000021615 conjugation Effects 0.000 description 1
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 230000001186 cumulative effect Effects 0.000 description 1
- 108010069495 cysteinyltyrosine Proteins 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000003748 differential diagnosis Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- 235000019301 disodium ethylene diamine tetraacetate Nutrition 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 229910000397 disodium phosphate Inorganic materials 0.000 description 1
- 235000019800 disodium phosphate Nutrition 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 239000012149 elution buffer Substances 0.000 description 1
- 238000004945 emulsification Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 201000006585 gastric adenocarcinoma Diseases 0.000 description 1
- 238000005227 gel permeation chromatography Methods 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
- 108010010147 glycylglutamine Proteins 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 210000002443 helper t lymphocyte Anatomy 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 238000003119 immunoblot Methods 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 108010009298 lysylglutamic acid Proteins 0.000 description 1
- 108010064235 lysylglycine Proteins 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000005374 membrane filtration Methods 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 238000011587 new zealand white rabbit Methods 0.000 description 1
- 239000011022 opal Substances 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 210000004332 phalangeal cell Anatomy 0.000 description 1
- 108010018625 phenylalanylarginine Proteins 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 108010077112 prolyl-proline Proteins 0.000 description 1
- 108010031719 prolyl-serine Proteins 0.000 description 1
- 229960000856 protein c Drugs 0.000 description 1
- 238000000197 pyrolysis Methods 0.000 description 1
- 230000036647 reaction Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 108010048818 seryl-histidine Proteins 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 235000017557 sodium bicarbonate Nutrition 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 108010061238 threonyl-glycine Proteins 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 239000000439 tumor marker Substances 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4747—Apoptosis related proteins
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/765—Serum albumin, e.g. HSA
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/77—Ovalbumin
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/795—Porphyrin- or corrin-ring-containing peptides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
-
- 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
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
-
- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
-
- 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
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Engineering & Computer Science (AREA)
- Gastroenterology & Hepatology (AREA)
- Immunology (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Zoology (AREA)
- Toxicology (AREA)
- Biotechnology (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Food Science & Technology (AREA)
- Cell Biology (AREA)
- Oncology (AREA)
- Hospice & Palliative Care (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a CIDE3 polypeptide, preparation of an antibody thereof and applications of the antibody and belongs to the field of bio-genetic engineering and medical. The amino acid sequence of the CIDE3 polypeptide is shown as SEQIDNO.1. The antibody prepared by adopting the sequence as an antigen can achieve specific recognition of a human CIDE3 protein. By a large amount of analysis, antigenicity, hydrophilicity and surface accessibility of the sequence shown as the SEQIDNO.1 are the strongest, so that the polyclonal antibody provided by the invention has good specific recognition effects for the CIDE3 protein and is stale in performance, high in sensitivity and strong in specificity. The polyclonal antibody can be used for specific recognition and detection of the human CIDE3 protein, and is expected to provide help for liver cancer diagnosis and to guide clinical prognosis and selection of therapeutic schedules.
Description
Technical field
The invention belongs to biological gene engineering and medical field.More specifically, the Synthesis and applications of a kind of CIDE3 polypeptide and antibody thereof is related to.
Background technology
DNA fragmentation factor (DNA Fragmentation Factor, DFF) participates in the important protein factor of an apoptotic class, is by two of 40KD and 45KD heterodimers that subunit is formed.Necrocytosis incitant (Cell death activator, CIDE) is the special DNA fragmentation factor (DFF) of a class.The N section homology of CIDEs albumen and DFF 45KD subunit, promotes apoptotic generation.In now known CIDE albumen, the mankind have CIDE-A, CIDE-B and CIDE3.Wherein, CIDE3 gene found in 2003, and be positioned on mankind 3p25 karyomit(e), total length about 169 kb, its cDNA contains 1305 bases, there is alternative splicing.
People CIDE3 albumen is present in primary hepatocarcinoma HCC(10/37 example), adenocarcinoma of stomach (4/19 example) and mammary cancer (1/17 is routine), in HCC cell, the positive expression rate (10/37 example) of people CIDE3 albumen is significantly lower than the other liver cell of cancer (28/29 example) (P<0.05), and wherein the positive expression rate of the middle people CIDE3 albumen of each differential period HCC cell (high, medium and low differentiation) is respectively lower than the other liver cell (P<0.05) of cancer.Because people CIDE3 gene is apoptosis-related genes, and apoptosis with tumour generation and develop closely related, these results prompter CIDE3 gene has certain dependency with tumour.Particularly in HCC, the positive expression rate of people CIDE3 albumen is starkly lower than the other liver cell of cancer, infers it is likely that the expression deletion of people CIDE3 albumen causes apoptosis function obstacle or plays synergy to the generation and the development that make tumour with other genes.In sum, there is very strong dependency, point out us between CIDE3 gene and liver cancer, CIDE3 can be used as a kind of mark of liver cancer.
Patent 200610041899.7 discloses the anti-polyclonal antibody of a kind of Human cell death-inducing DFF45-like effector-3 albumen rabbit, with Human cell death-inducing DFF45-like effector-3 albumen n end 172 amino acid for antigen immune New Zealand white rabbit obtains.This antibody can be used for molecular biology and the histology experiment research of CIDE-3 function, but it is longer that it still exists polypeptide fragment, needs recombinant expressed, the problems such as complicated operation is consuming time.Therefore, we are also not content with this, and the mark of further research and probe CIDE-3 albumen is a far reaching research topic.
Summary of the invention
Technical problem to be solved by this invention is the limitation and the deficiency that overcome existing CIDE3 labeling technique, provides the Synthesis and applications of a kind of CIDE3 polypeptide and antibody thereof.This CIDE3 polypeptide antibody can specific recognition people liver-neoplasm marker CIDE3, can be used for the detection of liver cancer marker CIDE3, and then for Diagnosis of Liver Neoplasm, differential diagnosis, predicting tumors transfer and prognosis.
The object of this invention is to provide a kind of CIDE3 polypeptide.
Another object of the present invention is to provide a kind of polyclonal antibody of CIDE3 polypeptide.
Still a further object of the present invention is to provide above-mentioned CIDE3 polypeptide and antibody is applied in specific marker people liver-neoplasm marker CIDE3.
Above-mentioned purpose of the present invention is achieved by the following technical programs:
The invention provides a kind of CIDE3 peptide sequence, its aminoacid sequence is as shown in SEQ ID NO.1.Shown in SEQ ID NO.1, sequence is: C-ATEEGQPPKGKASSL.
Present invention also offers a kind of coupling protein antigen, obtained by sequence shown in SEQ ID NO.1 and carrier protein couplet.
Preferably, described carrier proteins is keyhole limpet hemocyanin (keyhole limpet hemacyanin, KLH), bovine serum albumin (bovine serum albumin, BSA), ovalbumin (ovalbumin, or bovine thyroglobulin (bovine thyroglobulin, THY) OVA).
More preferably, described carrier proteins is keyhole limpet hemocyanin.
In addition, present invention also offers a kind of polyclonal antibody, with above-mentioned coupling protein antigen for antigen prepares.
Preferably, this polyclonal antibody prepares with above-mentioned coupling protein antigen immune rabbit.
The present invention also provides the application of above-mentioned polyclonal antibody in mark liver-neoplasm marker CIDE3.
The present invention obtains a kind of people CIDE3 aminoacid sequence NP_001186481.1 from GenBank database, is that a kind of fat drips albumen, is made up of (as shown in SEQ ID NO.2), protein molecular quality 26.8KD 238 amino acid.
Shown in SEQ ID NO.2, sequence is as follows:
MEYAMKSLSLLYPKSLSRHVSVRTSVVTQQLLSEPSPKAPRARPCRVSTADRSVRKGIMAYSLEDLLLKVRDTLMLADKPFFLVLEEDGTTVETEEYFQALAGDTVFMVLQKGQKWQPPSEQGTRHPLSLSHKPAKKIDVARVTFDLYKLNPQDFIGCLNVKATFYDTYSLSYDLHCCGAKRIMKEAFRWALFSMQATGHVLLGTSCYLQQLLDATEEGQPPKGKASSLIPTCLKILQ。
The secondary structure, immunogenicity, hydrophilic and hydrophobic, surperficial accessibility etc. of the present invention to above-mentioned CIDE3 protein amino acid sequence are analyzed, and determine one section of suitable peptide sequence and carry out synthetic, obtain peptide sequence shown in SEQ ID NO.1; By the carrier mcKLH coupling of this peptide sequence and maleimide activation, this coupled product is carried out immunize New Zealand rabbit after desalting column purifying; Rabbit anteserum ELISA method antagonist through repeatedly immunity is tired and is detected, and tires after reaching ideal value and collects immunize rabbit serum, and with CIDE3 antigen peptide affinity purification column purification antibody; Purified antibodies is carried out to the qualifications such as ELISA, Western Bot.Qualification result shows, this polyclonal antibody can specific recognition CIDE3 albumen, can be used for detecting the CIDE3 albumen that liver cancer cell is expressed, and for diagnosing cancer of liver is offered help, and its clinical prognosis can be instructed to judge and the selection for the treatment of plan.
And the polyclonal antibody of anti-CIDE3 provided by the invention is with sequence shown in SEQ ID NO.1 for antigen prepares, 15 amino acid of sequence shown in SEQ ID NO.1 and the nearly C-terminal of people CIDE3 albumen.The present invention is through large component analysis, and the antigenicity of sequence shown in this SEQ ID NO.1, wetting ability and surperficial accessibility are the strongest, and are positioned at PROTEIN C end, more easily expose and identify.Therefore, the specific recognition of polyclonal antibody provided by the invention to CIDE3 albumen is effective, stable performance, and susceptibility is high, high specificity.
The present invention has following beneficial effect:
The present invention have selected CIDE3 albumen nearly C-terminal 15 peptide alternatively polypeptide, and adopt manual method to carry out Peptide systhesis to prepare immunogen, further immunize New Zealand rabbit obtains the polyclonal antibody of anti-CIDE3, obtains the monospecific rabbit polyclonal antibody of anti-CIDE3 finally by antigen peptide affinity purification method.This monospecific rabbit polyclonal antibody can specific recognition CIDE3 albumen, can be used for detecting the CIDE3 albumen that liver tumor cells is expressed, and for diagnosing cancer of liver is offered help, and its clinical prognosis can be instructed to judge and the selection for the treatment of plan.
In addition, the present invention adopts C-terminal small peptide as antigen, and can direct labor synthesize, do not need recombinant expressed, prepare efficient and convenient, with low cost, the cycle is short.In addition, this epitope is clear and definite, and is positioned at C-terminal and more easily exposes, and the rabbit antibody obtained with this antigen-immunized animal has the characteristic of monoclonal antibody, very easily epitope in specific recognition clinical sample.
Accompanying drawing explanation
Fig. 1 adopts DNAstar software analysis people CIDE3 protein characteristic.The sequence marked in frame is selected peptide sequence, and this peptide sequence is positioned at the nearly C-terminal of CIDE3 protein, and its antigenicity, wetting ability and surperficial accessibility are stronger.
Fig. 2 be anti-CIDE3 polyclonal antibody after antigen peptide affinity purification through SDS-PAGE electrophoresis (12%) detected result.M is albumen Marker; Lane 1 is loading peak; Lane 2 wears peak for stream; Lane 3 is balance peak; Lane 4 is elution peak.
Fig. 3 is the Western-Blot qualification result of the anti-CIDE3 polyclonal antibody in the present invention.Loading is HepG2 cell (Bel7402) lysate.
The immunodotting qualification of the anti-CIDE3 polyclonal antibody in Fig. 4 the present invention.
Fig. 5 implementing procedure of the present invention.
Embodiment
Further illustrate the present invention below in conjunction with Figure of description and specific embodiment, but embodiment does not limit in any form to the present invention.Under the prerequisite not deviating from the technical scheme that the present invention deals with problems, any change of those of ordinary skill in the art's easy realization made for the present invention all will fall within right of the present invention.
Unless stated otherwise, the present invention adopts reagent, method and apparatus are the art conventional reagent, method and apparatus.Unless stated otherwise, agents useful for same of the present invention and material are commercial.
the design of embodiment 1 CIDE3 peptide sequence
1, CIDE3 aminoacid sequence
The present invention obtains a kind of people CIDE3 aminoacid sequence NP_001186481.1 from GenBank database, is made up of (as shown in SEQ ID NO.2), protein molecular quality 26.8KD 238 amino acid.
Shown in SEQ ID NO.2, sequence is as follows:
MEYAMKSLSLLYPKSLSRHVSVRTSVVTQQLLSEPSPKAPRARPCRVSTADRSVRKGIMAYSLEDLLLKVRDTLMLADKPFFLVLEEDGTTVETEEYFQALAGDTVFMVLQKGQKWQPPSEQGTRHPLSLSHKPAKKIDVARVTFDLYKLNPQDFIGCLNVKATFYDTYSLSYDLHCCGAKRIMKEAFRWALFSMQATGHVLLGTSCYLQQLLDATEEGQPPKGKASSLIPTCLKILQ。
2, http://www.uniprot.org/uniprot on-line analysis CIDE3 structural domain.
Analytical results (as shown in table 1) shows, and people CIDE3 albumen contains 1 structural domain, namely.
Table 1
Wherein, aa represents amino acid, and Chain represents DNA chain, and Domain represents protein structure domain, and Cell death activator CIDE-3 is exactly phalangeal cell apoptosis activated protein c IDE-3.
3, with DNAstar software analysis CIDE3 protein characteristic, analytical results is as shown in table 2.People CIDE3 molecular weight of albumen is 26.753KD, and iso-electric point is 8.75, is basic protein.
Table 2
Wherein, the translation of each word is: Analysis: analyze; Molecular Weight: molecular weight; Length: length; Molar Extinction Coefficient: molar extinction coefficient; Isoelectric Point: iso-electric point (pl); Charge at pH 7: the electric charge under pH7 condition; Whole Protein: whole protein; PMoles and unit pmol.
4, by DNAstar software analysis CIDE3 immunogenicity, hydrophilic and hydrophobic and surperficial accessibility, result as shown in Figure 1.The nearly C-terminal of people CIDE3 albumen has 15 amino acid antigenicities, wetting ability and surperficial accessibility stronger.Therefore, the peptide sequence that the present invention selects is ATEEGQPPKGKASSL(215aa ~ 229aa).
the synthesis of embodiment 2 coupling protein antigen
1, Peptide systhesis
For ease of with carrier protein couplet, during improvement on synthesis N end add halfcystine, that is: a C-ATEEGQPPKGKASSL.Polypeptide is synthesized by gill biochemical corp, Shanghai.
2, polypeptide and carrier protein couplet
The polypeptide antigen of chemosynthesis is small molecules, itself is difficult to the antigenicity had, and can only produce very weak immune response by induced animal, thus itself and carrier proteins are cross-linked by the present invention.Carrier proteins contains a lot of epitope, can stimulate t helper cell, and then elicit B cell reaction.
Have multiple for the carrier proteins crosslinked with polypeptide, the carrier wherein the most generally used is keyhole limpet hemocyanin (keyhole limpet hemacyanin, KLH), bovine serum albumin (bovine serum albumin, BSA), ovalbumin (ovalbumin, and bovine thyroglobulin (bovine thyroglobulin, THY) OVA).Wherein, KLH and BSA is conventional, but is also often used as the blocker of detection experiment due to BSA, and the antibody that the method is produced also exists certain limitation in application.KLH of the present invention has higher antigenicity.
3, polypeptide and carrier protein couplet:
(1) dilute the mcKLH of a pipe maleimide activation with 200 μ L ultrapure waters, be diluted to the solution of 10 mg/mL.(note: above-mentioned solution is that pearl opal is translucent, must not vibrate or heat, otherwise mcKLH can be caused to precipitate.）
(2) haptens containing sulfydryl is dissolved with the coupling Buffer being equivalent to solution 1.0 ~ 2.5 times of volumes in step (1).Such as: 2 mg haptens, 200 ~ 500 μ L coupling Buffer are dissolved, joins mcKLH.
(note: if haptens polypeptide is Yi Rong, can join in mcKLH suspension in solid form.If haptens is not easily molten, can add DMSO increases dissolving.DMSO in coupling lysate concentration lower than 30%, otherwise carrier proteins volatility.）
(3) polypeptide, mcKLH is mixed immediately, then at room temperature reaction 2h.
4, coupled product purifying: carry out purifying conjugate by gel chromatography desalting column
If conjugate was injected within one week, purify with PBS.If conjugate is frozen, purify with purifying buffering salt, if form precipitation when coupling, centrifugal, collect supernatant, retain precipitation, only purifying supernatant.The conjugate of purifying and precipitation are combined.
Purification step is as follows:
(1) dissolve one bottle of purifying buffering salt, add the ultrapure water that 60mL is degassed.4 DEG C of preservations.
(2) to take away the lid of T&B of desalting column, storage liquid is discharged.One desalting column can purifying 0.5mL sample.
(3) pillar is rinsed with the purification buffer of 3 ~ 5 times of column volumes (15 ~ 25mL).
D. the peptide carrier mixture of 0.5ml is directly added post center.Add the purification buffer of 0.5ml, in separator tube, collect each peak.
E. under 280nm, absorbancy is measured to determine which part is containing conjugate.What detect at first absorption peak will be hapten conjugation thing.Mix all parts containing conjugate
F. after the part containing conjugate occurs, continue to add damping fluid in post, collect the haptens not having coupling.
G. by conjugate filtration sterilization, Preservation in sterile condition is at-80 DEG C.
5, result: coomassie brilliant blue records protein concentration and content after coupling, often pipe 250 μ g packing ,-80 DEG C of preservations.
the preparation of the anti-CIDE3 rabbit polyclonal antibody of embodiment 3 and purifying
1, animal immune
Often kind of coupled product immunity two new zealand male rabbits, body weight 2 ~ 2.5kg.Not formula Freund's complete adjuvant and Fu Shi Freund's incomplete adjuvant available from Sigma.
Immune programme for children (as shown in table 3):
(1) dilute 100 μ g antigens to 1.25mL with PBS, with the mixing of equal-volume adjuvant, vortex vibrates 100 minutes, detects emulsification result, gets an antigen and drip in waterborne, insoluble solution diffusion in a minute.
(2) immune vestibule venous blood sampling, 4 DEG C leave standstill after get serum ,-80 DEG C of preservations, make negative control sera.
Table 3
2, antibody titer ELISA detection method
(1) experiment reagent
Phosphate buffered saline buffer (PBS) (10 × concentrated), sodium-chlor (NaCl) 50g, Repone K (KCl) 1.25g, potassium primary phosphate (KH
2pO
4) 1.25g, Sodium phosphate dibasic (Na
2hPO
412H
2o) 18.1g, distilled water adds to 1000mL, adjusts pH to 7.2 with 1M HCl.
Confining liquid: normal calf serum 10mL, 1 × PBS(be not containing tween) 90mL.
Lavation buffer solution: tween 20 (Tween 20) 0.2mL, 1 × PBS 1000mL.
Substrate colour developing A liquid: sodium-acetate (CH
3cOONa) 13.6g, citric acid (C
6h
8o
7h
2o) 1.6g, hydrogen peroxide (H
2o
2, 30%) and 0.3mL, distilled water adds to 500mL.
Substrate colour developing B liquid: disodium ethylene diamine tetraacetate (EDTA-Na
2) 0.2g, citric acid (C
6h
8o
7h
2o) 0.95g, glycerine (C
3h
8o
3) 50mL, tetramethyl benzidine (TMB) 0.2g, distilled water adds to 500mL.
Stop buffer (2M H
2sO
4): get dense H
2sO
427.62mL, slowly joins in the distilled water of 473mL, mixes.
Antigen coated liquid (0.1M carbonate buffer solution) pH 9.6: sodium carbonate (Na
2cO
3) 1.59g, sodium bicarbonate (NaHCO
3) 2.93g, sodium azide (NaN
3) 0.2g, distilled water adds to 1000mL.
The goat anti-mouse igg (commercialization) of horseradish peroxidase-labeled.
(2) experimental procedure
S1. antigen coated: pure polypeptide antigen final concentration is generally 1 ~ 2 μ g/mL, get 100 μ L add in each hole of polystyrene enzyme joint inspection drafting board with after coating buffer dilution, 4 DEG C spend the night after, wash liquid 3 times.Advise that 4 DEG C of bags are spent the night.
S2. close: every hole adds 200 μ L or fills it up with confining liquid, 4 DEG C are spent the night or 37 DEG C after two hours, wash 3 times, pat dry.Put 4 DEG C of Refrigerator stores for subsequent use.
S3.ELISA detects
S31. add testing sample: separation of serum after rabbit ear vein blood sampling, use PBS doubling dilution, 50 ~ 100 μ L/ holes are loaded onto bag by good enzyme plate, and meanwhile, choosing the front rabbit anteserum of immunity is respectively negative control, hatches 30min for 37 DEG C, washs 3 times, pat dry.
S32. add two to resist: according to the selection extension rate of tiring of ELIAS secondary antibody, 100 μ L/ holes, hatch 30min for 37 DEG C, wash 3 times, pat dry.
S33. develop the color: add each 80 μ L/holes of A, B liquid, 37 DEG C of colour developing 15min.
S34. stop: add stop buffer 80 μ L/hole.
S35. reading: measure each hole OD value with 450nm Single wavelength, is limited to be greater than 2.5 with the ratio (P/N) of negative control hole OD value, as being judged as the positive or determining the stagnation point of tiring.
3, rabbit anteserum is collected
Adopt strength arterial blood letting method, non-anti-freezing rabbit whole blood hatches 2h at 37 DEG C after collecting.
4, antibody purification:
With CIDE3 antigen peptide bag by NHS activated sepharose (being purchased from GE company), step is as follows:
(1) the swelling 1 g NHS activated sepharose dry powder of ice-cold 1 mM HCl, washs four times with the 1 mM HCl of cumulative volume 200 mL, each 50 mL.
(2) 10 mg CIDE3 antigen peptide are dissolved in 4 mL bags and are buffered liquid (containing 0.1 M NaHCO
3, pH 8.3,0.5 M NaCl) in.
(3) shift solution immediately in the NHS activated sepharose of activation, turn upside down mixing incubated at room 2h.
(4) with 0.2 M glycine, pH 8.0 room temperature closes coupling agarose gel 2h.
(5) with 0.1 M sodium acetate, 0.5 M NaCl, pH 4.0 solution and bag are buffered liquid and alternately wash coupling agarose gel 4 to 5 times.
In immunize rabbit serum, the affinity purification step of antigen-specific CIDE3 antibody is as follows:
After (1) 20 mL rabbit anteserum 0.01M PBS carries out 5 times of dilutions, by loading after 0.45 μm of membrane filtration.
(2) with elution buffer (0.1 M glycine pH=2.7) wash-out target protein, and add neutralization buffer (1 M Tris-HCl pH=9.0) and make antibody neutralize neutral left and right (pH=7.0-8.0), then purifying protein is dialysed with PBS immediately.
(3), after concentrated with ultrafiltration cup, protein concentration is measured.Be stored in 4 DEG C of refrigerators for subsequent use.The anti-CIDE3 antibody of final purification is through SDS-PAGE preliminary evaluation.
Result shows, and, it is 1.3mg/mL that ultraviolet spectrophotometer measures antibody concentration after antibody purification through SDS-PAGE protein electrophoresis qualification result as shown in Figure 2.Purified antibodies is stored in-20 DEG C, in 0.01M PBS damping fluid, containing 40% glycerine and 0.5% BSA in solution.
the qualification of the anti-CIDE3 polyclonal antibody of embodiment 4
1, the ELISA qualification of anti-CIDE3 polyclonal antibody
With the CIDE3 polypeptide of synthesis for detectable antigens, coated elisa plate, using the not immune rabbit anteserum of 1:2000 dilution as negative control, by the antibody doubling dilution after rabbit anteserum and purifying, application ELISA method detects, and is judged as the positive to be greater than 2.1 with the ratio of negative serum, calculates monoclonal antibody and tires.
Result shows, and it is as shown in table 4 that the tiring of the anti-CIDE3 antibody of purifying reaches 1:16000().
The anti-CIDE3 antibody titer of table 4
2, the Western-Blot qualification of anti-CIDE3 polyclonal antibody
Collect HepG2 cell 1 × 10
7individual, after its cracking, lysate carries out 12% SDS-PAGE electrophoresis, with reference to the western blotting method described in " molecular cloning ", electrotransfer condition is 100 V electrophoresis 1 h, and confining liquid is the TBST containing 5% skim-milk, and primary antibodie is the CIDE3 polyclonal antibody (final concentration 1.3 mg/mL) in the present invention, two resist the sheep anti-mouse igg antibody for horseradish peroxidase-labeled, carry out immunoblotting assay by ECL Color Appearance System.
Result shows, and anti-CIDE3 polyclonal antibody of the present invention can detect the protein band about 26.8KD size in HepG2 cell pyrolysis liquid, conforms to (as shown in Figure 3) with people CIDE3 molecular weight of albumen size.
3, the immunodotting qualification of anti-CIDE3 polyclonal antibody, CIDE3 polypeptide 10 μ g point pvdf membrane, the anti-CIDE3 of rabbit is many, and anti-3 μ g/mL are hatched.
Result as shown in Figure 4.
In addition, the implementing procedure that the present invention makes a search as shown in Figure 5 for qualification NG8.
SEQUENCE LISTING
<110> Chinese Academy of Sciences Guangzhou Institute of Biomedicine and Health
The Synthesis and applications of <120> CIDE3 polypeptide and antibody thereof
<130>
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 16
<212> PRT
<213> CIDE3 peptide sequence
<400> 1
Cys Ala Thr Glu Glu Gly Gln Pro Pro Lys Gly Lys Ala Ser Ser Leu
1 5 10 15
<210> 2
<211> 238
<212> PRT
<213> CIDE3 protein amino acid sequence
<400> 2
Met Glu Tyr Ala Met Lys Ser Leu Ser Leu Leu Tyr Pro Lys Ser Leu
1 5 10 15
Ser Arg His Val Ser Val Arg Thr Ser Val Val Thr Gln Gln Leu Leu
20 25 30
Ser Glu Pro Ser Pro Lys Ala Pro Arg Ala Arg Pro Cys Arg Val Ser
35 40 45
Thr Ala Asp Arg Ser Val Arg Lys Gly Ile Met Ala Tyr Ser Leu Glu
50 55 60
Asp Leu Leu Leu Lys Val Arg Asp Thr Leu Met Leu Ala Asp Lys Pro
65 70 75 80
Phe Phe Leu Val Leu Glu Glu Asp Gly Thr Thr Val Glu Thr Glu Glu
85 90 95
Tyr Phe Gln Ala Leu Ala Gly Asp Thr Val Phe Met Val Leu Gln Lys
100 105 110
Gly Gln Lys Trp Gln Pro Pro Ser Glu Gln Gly Thr Arg His Pro Leu
115 120 125
Ser Leu Ser His Lys Pro Ala Lys Lys Ile Asp Val Ala Arg Val Thr
130 135 140
Phe Asp Leu Tyr Lys Leu Asn Pro Gln Asp Phe Ile Gly Cys Leu Asn
145 150 155 160
Val Lys Ala Thr Phe Tyr Asp Thr Tyr Ser Leu Ser Tyr Asp Leu His
165 170 175
Cys Cys Gly Ala Lys Arg Ile Met Lys Glu Ala Phe Arg Trp Ala Leu
180 185 190
Phe Ser Met Gln Ala Thr Gly His Val Leu Leu Gly Thr Ser Cys Tyr
195 200 205
Leu Gln Gln Leu Leu Asp Ala Thr Glu Glu Gly Gln Pro Pro Lys Gly
210 215 220
Lys Ala Ser Ser Leu Ile Pro Thr Cys Leu Lys Ile Leu Gln
225 230 235
Claims (7)
1. a CIDE3 peptide sequence, is characterized in that, its aminoacid sequence is as shown in SEQ ID NO.1.
2. a coupling protein antigen, is characterized in that, is to be obtained by sequence shown in SEQ ID NO.1 and carrier protein couplet.
3. coupling protein antigen according to claim 2, it is characterized in that, described carrier proteins is keyhole limpet hemocyanin, bovine serum albumin, ovalbumin or bovine thyroglobulin.
4. coupling protein antigen according to claim 2, it is characterized in that, described carrier proteins is keyhole limpet hemocyanin.
5. a polyclonal antibody, is characterized in that, with coupling protein antigen described in claim 2 for antigen prepares.
6. polyclonal antibody according to claim 5, is characterized in that, prepare with coupling protein antigen immune rabbit described in claim 2.
7. the application of polyclonal antibody described in claim 5 in mark liver-neoplasm marker CIDE3.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410391896.0A CN104311654A (en) | 2014-08-11 | 2014-08-11 | CIDE3 polypeptide, preparation of antibody thereof and applications of the antibody |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410391896.0A CN104311654A (en) | 2014-08-11 | 2014-08-11 | CIDE3 polypeptide, preparation of antibody thereof and applications of the antibody |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN104311654A true CN104311654A (en) | 2015-01-28 |
Family
ID=52366989
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410391896.0A Pending CN104311654A (en) | 2014-08-11 | 2014-08-11 | CIDE3 polypeptide, preparation of antibody thereof and applications of the antibody |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104311654A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110913885A (en) * | 2017-06-15 | 2020-03-24 | 俄亥俄大学 | Methods of modulating free fatty acid flux using fat specific protein 27(FSP27) compositions |
| CN113121683A (en) * | 2021-04-14 | 2021-07-16 | 南通大学 | Preparation method of cotton GraiRGA transcription factor specific recognition antibody |
| CN113683674A (en) * | 2021-10-14 | 2021-11-23 | 山东省淡水渔业研究院（山东省淡水渔业监测中心） | Epitope polypeptide of anti-snakehead Nramp antibody and application of epitope polypeptide in antibody preparation |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1388132A (en) * | 2001-05-25 | 2003-01-01 | 中国科学院上海生物化学研究所 | Cell death inducing protein and its coding sequence and use |
| CN1821404A (en) * | 2006-03-09 | 2006-08-23 | 中国人民解放军第四军医大学 | Rabbit anti-human CIDE-3 protein poly clonal antibody |
-
2014
- 2014-08-11 CN CN201410391896.0A patent/CN104311654A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1388132A (en) * | 2001-05-25 | 2003-01-01 | 中国科学院上海生物化学研究所 | Cell death inducing protein and its coding sequence and use |
| CN1821404A (en) * | 2006-03-09 | 2006-08-23 | 中国人民解放军第四军医大学 | Rabbit anti-human CIDE-3 protein poly clonal antibody |
Non-Patent Citations (3)
| Title |
|---|
| NCBI: "Cell death activator CIDE-3 isoform 3[Homo sapiens]", 《GENBANK》 * |
| 胡小元: "用合成多肽为半抗原制备Bt Cry1Ab的单克隆抗体", 《农业生物技术学报》 * |
| 闵婕: "CIDE-3对肝癌细胞SMMC-7721凋亡影响及其相互作用分子在酵母双杂交系统中的筛选", 《中国博士学位论文全文数据库》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110913885A (en) * | 2017-06-15 | 2020-03-24 | 俄亥俄大学 | Methods of modulating free fatty acid flux using fat specific protein 27(FSP27) compositions |
| CN113121683A (en) * | 2021-04-14 | 2021-07-16 | 南通大学 | Preparation method of cotton GraiRGA transcription factor specific recognition antibody |
| CN113121683B (en) * | 2021-04-14 | 2022-01-14 | 南通大学 | Preparation method of cotton GraiRGA transcription factor specific recognition antibody |
| CN113683674A (en) * | 2021-10-14 | 2021-11-23 | 山东省淡水渔业研究院（山东省淡水渔业监测中心） | Epitope polypeptide of anti-snakehead Nramp antibody and application of epitope polypeptide in antibody preparation |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102633864B (en) | GPC3 antigen polypeptide, anti-GPC3 polyclonal antibody and application thereof | |
| AU2010321790B2 (en) | Peptides, devices, and methods for the detection of Ehrlichia antibodies | |
| CN101525614A (en) | Human oophoroma tumor marker HE4 enzymoimmunoassay kit | |
| FR2502154A1 (en) | PROCESS FOR PREPARING ANTIGENS FOR VIRAL HEPATITIS NANB, AND APPLICATION TO THE REAGENT FOR THE DIAGNOSIS AND PROGNOSIS OF INFECTIONS CAUSED BY VIRAL HEPATITIS VIRUSES | |
| CN113683702B (en) | Preparation method and application of polyclonal antibody of striped bamboo shark single-domain antibody | |
| BR112015008036B1 (en) | POPULATION OF ISOLATED PEPTIDES, METHOD TO DETECT AN ANTIBODY IN A SAMPLE, METHOD TO DIAGNOSE MONOCYTIC AND/OR GRANULOCYTIC EHRLICHIOSIS, METHOD TO DETECT THE PRESENCE OF ANTIBODIES AND KIT | |
| KR20180083941A (en) | Mutant HEV polypeptides and their uses for assaying anti-HEV antibodies | |
| CN104311654A (en) | CIDE3 polypeptide, preparation of antibody thereof and applications of the antibody | |
| NO303852B1 (en) | Procedure for detecting analyte variants, as well as kits for performing the method | |
| CN102336814B (en) | Polypeptide sequence for preparing anti-liver-neoplasm marker CK-19 antibody, polyclonal antibody and application thereof | |
| ES2205593T3 (en) | ANTIBODIES AGAINST VASP (PHOSPHOPROTEIN STIMULATED BY VASODILATORS), HYBRIDID CELLS FOR PREPARATION, AND THE USE OF SUCH ANTIBODIES. | |
| CN104744580B (en) | A kind of TgVP1 extracellular regions antigen polypeptide, the polyclonal antibody of anti-TgVP1 and its application | |
| ES2247633T3 (en) | METHODS TO DETERMINE THE PRESENCE OF THE S-100 BETA BRAIN PROTEIN. | |
| CN110317270A (en) | Antitoxin snake PLA2Protein antibodies and its application | |
| Dmitriev et al. | Western blot analysis of human and rat serotonin transporter in platelets and brain using site-specific antibodies: evidence that transporter undergoes endoproteolytic cleavage | |
| ES2345569T3 (en) | PEPTIDO APTAMERO TO NEUTRALIZE THE UNION OF SPECIFIC ANTIBODIES TO ANTIGEN PLATELETS AND THERAPEUTIC AND DIAGNOSTIC APPLICATIONS CONTAINING IT. | |
| CN105296520B (en) | A kind of preparation method and its enzyme-linked immunologic detecting kit of human tumor antigen 3H11Ag | |
| CN103102392B (en) | N-terminal polypeptide of retinoic acid induced protein 16 and preparation method and application of antibody thereof | |
| CN109293762A (en) | DRC3f antigen polypeptide, the polyclonal antibody of anti-DRC3f and application | |
| CN104744581B (en) | A kind of toxoplasma protein TgVP1 intracellular regions antigen polypeptide, the polyclonal antibody of resisting toxoplasmosis TgVP1 and its application | |
| JPS62246596A (en) | Modified beta 2 microglobulin | |
| ES2334972T3 (en) | IMMUNOENSAY FOR HUMAN MEDULASINE AND DIAGNOSTIC METHOD OF MULTIPLE SCLEROSIS. | |
| CN108623662A (en) | Phytopathogen antigenic compound, antibody, detection kit and its application | |
| Akhidova et al. | Antibodies to synthetic peptides for the detection of survivin in tumor tissues | |
| CN112903996A (en) | nCoV-N protein detection kit and nCoV-N protein detection method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20150128 |