CN112574306A - 脂联素单克隆抗体、抗体对及其制备方法和用途 - Google Patents
脂联素单克隆抗体、抗体对及其制备方法和用途 Download PDFInfo
- Publication number
- CN112574306A CN112574306A CN202011504233.7A CN202011504233A CN112574306A CN 112574306 A CN112574306 A CN 112574306A CN 202011504233 A CN202011504233 A CN 202011504233A CN 112574306 A CN112574306 A CN 112574306A
- Authority
- CN
- China
- Prior art keywords
- gly
- ala
- thr
- cys
- adiponectin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 108010076365 Adiponectin Proteins 0.000 title claims abstract description 119
- 238000002360 preparation method Methods 0.000 title claims abstract description 26
- 102000011690 Adiponectin Human genes 0.000 title abstract description 89
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 38
- 238000000034 method Methods 0.000 claims abstract description 21
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 9
- 239000004816 latex Substances 0.000 claims abstract description 9
- 229920000126 latex Polymers 0.000 claims abstract description 9
- 238000003018 immunoassay Methods 0.000 claims abstract description 6
- 239000003814 drug Substances 0.000 claims abstract description 5
- 210000004027 cell Anatomy 0.000 claims description 143
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 60
- 238000012258 culturing Methods 0.000 claims description 50
- 239000001963 growth medium Substances 0.000 claims description 41
- 239000007788 liquid Substances 0.000 claims description 31
- 230000003053 immunization Effects 0.000 claims description 29
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 claims description 27
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 claims description 27
- 210000004989 spleen cell Anatomy 0.000 claims description 27
- 102000004169 proteins and genes Human genes 0.000 claims description 25
- 239000006228 supernatant Substances 0.000 claims description 25
- 241000699666 Mus <mouse, genus> Species 0.000 claims description 24
- 210000004408 hybridoma Anatomy 0.000 claims description 23
- 230000001580 bacterial effect Effects 0.000 claims description 21
- 238000001514 detection method Methods 0.000 claims description 19
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 18
- 241001465754 Metazoa Species 0.000 claims description 17
- 238000002649 immunization Methods 0.000 claims description 16
- 239000013612 plasmid Substances 0.000 claims description 16
- 230000004927 fusion Effects 0.000 claims description 15
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 claims description 15
- 238000002156 mixing Methods 0.000 claims description 13
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 12
- 238000004113 cell culture Methods 0.000 claims description 12
- 230000006698 induction Effects 0.000 claims description 12
- 238000009630 liquid culture Methods 0.000 claims description 12
- 238000004519 manufacturing process Methods 0.000 claims description 12
- 239000000243 solution Substances 0.000 claims description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 12
- 239000006285 cell suspension Substances 0.000 claims description 11
- 239000000047 product Substances 0.000 claims description 10
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 9
- 239000012880 LB liquid culture medium Substances 0.000 claims description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 8
- 239000002671 adjuvant Substances 0.000 claims description 8
- 229960000723 ampicillin Drugs 0.000 claims description 8
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 claims description 8
- 238000003113 dilution method Methods 0.000 claims description 8
- 239000002609 medium Substances 0.000 claims description 8
- 238000000746 purification Methods 0.000 claims description 8
- 238000012163 sequencing technique Methods 0.000 claims description 8
- 210000002966 serum Anatomy 0.000 claims description 8
- 239000007787 solid Substances 0.000 claims description 8
- 230000009466 transformation Effects 0.000 claims description 8
- 230000001131 transforming effect Effects 0.000 claims description 8
- 230000007910 cell fusion Effects 0.000 claims description 7
- 238000011068 loading method Methods 0.000 claims description 7
- 239000011248 coating agent Substances 0.000 claims description 6
- 238000000576 coating method Methods 0.000 claims description 6
- 238000005406 washing Methods 0.000 claims description 6
- 101150096655 APM1 gene Proteins 0.000 claims description 5
- 238000001712 DNA sequencing Methods 0.000 claims description 5
- 102000004190 Enzymes Human genes 0.000 claims description 5
- 108090000790 Enzymes Proteins 0.000 claims description 5
- 241000699670 Mus sp. Species 0.000 claims description 5
- 238000007865 diluting Methods 0.000 claims description 5
- 230000008569 process Effects 0.000 claims description 5
- 238000012216 screening Methods 0.000 claims description 5
- 238000012360 testing method Methods 0.000 claims description 5
- 101150112998 ADIPOQ gene Proteins 0.000 claims description 4
- 229920000936 Agarose Polymers 0.000 claims description 4
- 101100162826 Dictyostelium discoideum apm2 gene Proteins 0.000 claims description 4
- 241000588724 Escherichia coli Species 0.000 claims description 4
- 101100053794 Homo sapiens ZBTB7C gene Proteins 0.000 claims description 4
- 238000012408 PCR amplification Methods 0.000 claims description 4
- 229920002534 Polyethylene Glycol 1450 Polymers 0.000 claims description 4
- 239000012980 RPMI-1640 medium Substances 0.000 claims description 4
- 210000000683 abdominal cavity Anatomy 0.000 claims description 4
- 238000010521 absorption reaction Methods 0.000 claims description 4
- 239000000654 additive Substances 0.000 claims description 4
- 238000001042 affinity chromatography Methods 0.000 claims description 4
- 238000000246 agarose gel electrophoresis Methods 0.000 claims description 4
- 238000004458 analytical method Methods 0.000 claims description 4
- 239000002518 antifoaming agent Substances 0.000 claims description 4
- 239000002299 complementary DNA Substances 0.000 claims description 4
- 239000003480 eluent Substances 0.000 claims description 4
- 238000001976 enzyme digestion Methods 0.000 claims description 4
- 238000011081 inoculation Methods 0.000 claims description 4
- 229930027917 kanamycin Natural products 0.000 claims description 4
- 229960000318 kanamycin Drugs 0.000 claims description 4
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 claims description 4
- 229930182823 kanamycin A Natural products 0.000 claims description 4
- 239000002245 particle Substances 0.000 claims description 4
- 238000001556 precipitation Methods 0.000 claims description 4
- 238000010839 reverse transcription Methods 0.000 claims description 4
- 238000003757 reverse transcription PCR Methods 0.000 claims description 4
- 101150116497 sacm1l gene Proteins 0.000 claims description 4
- 239000004017 serum-free culture medium Substances 0.000 claims description 4
- 239000011780 sodium chloride Substances 0.000 claims description 4
- 230000002194 synthesizing effect Effects 0.000 claims description 4
- 238000012795 verification Methods 0.000 claims description 4
- 239000007762 w/o emulsion Substances 0.000 claims description 4
- 241000894006 Bacteria Species 0.000 claims description 3
- 229930195731 calicheamicin Natural products 0.000 claims description 3
- HXCHCVDVKSCDHU-LULTVBGHSA-N calicheamicin Chemical compound C1[C@H](OC)[C@@H](NCC)CO[C@H]1O[C@H]1[C@H](O[C@@H]2C\3=C(NC(=O)OC)C(=O)C[C@](C/3=C/CSSSC)(O)C#C\C=C/C#C2)O[C@H](C)[C@@H](NO[C@@H]2O[C@H](C)[C@@H](SC(=O)C=3C(=C(OC)C(O[C@H]4[C@@H]([C@H](OC)[C@@H](O)[C@H](C)O4)O)=C(I)C=3C)OC)[C@@H](O)C2)[C@@H]1O HXCHCVDVKSCDHU-LULTVBGHSA-N 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 3
- 238000007710 freezing Methods 0.000 claims description 3
- 230000008014 freezing Effects 0.000 claims description 3
- 238000007912 intraperitoneal administration Methods 0.000 claims description 3
- 238000010079 rubber tapping Methods 0.000 claims description 3
- 102100031786 Adiponectin Human genes 0.000 claims 29
- 239000002994 raw material Substances 0.000 abstract description 4
- 238000005215 recombination Methods 0.000 abstract description 3
- 230000006798 recombination Effects 0.000 abstract description 3
- 108010061238 threonyl-glycine Proteins 0.000 description 45
- XKUKSGPZAADMRA-UHFFFAOYSA-N glycyl-glycyl-glycine Chemical compound NCC(=O)NCC(=O)NCC(O)=O XKUKSGPZAADMRA-UHFFFAOYSA-N 0.000 description 39
- 108010076324 alanyl-glycyl-glycine Proteins 0.000 description 36
- VPZXBVLAVMBEQI-UHFFFAOYSA-N glycyl-DL-alpha-alanine Natural products OC(=O)C(C)NC(=O)CN VPZXBVLAVMBEQI-UHFFFAOYSA-N 0.000 description 36
- ALNKNYKSZPSLBD-ZDLURKLDSA-N Cys-Thr-Gly Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(O)=O ALNKNYKSZPSLBD-ZDLURKLDSA-N 0.000 description 24
- 108010047495 alanylglycine Proteins 0.000 description 24
- 108010004073 cysteinylcysteine Proteins 0.000 description 24
- OBVSBEYOMDWLRJ-BFHQHQDPSA-N Ala-Gly-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@H](C)N OBVSBEYOMDWLRJ-BFHQHQDPSA-N 0.000 description 21
- KUFVXLQLDHJVOG-SHGPDSBTSA-N Ala-Thr-Thr Chemical compound C[C@H]([C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)O)NC(=O)[C@H](C)N)O KUFVXLQLDHJVOG-SHGPDSBTSA-N 0.000 description 21
- 108010079364 N-glycylalanine Proteins 0.000 description 21
- UZJDBCHMIQXLOQ-HEIBUPTGSA-N Thr-Cys-Thr Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CS)C(=O)N[C@@H]([C@@H](C)O)C(=O)O)N)O UZJDBCHMIQXLOQ-HEIBUPTGSA-N 0.000 description 21
- VGPWRRFOPXVGOH-BYPYZUCNSA-N Ala-Gly-Gly Chemical compound C[C@H](N)C(=O)NCC(=O)NCC(O)=O VGPWRRFOPXVGOH-BYPYZUCNSA-N 0.000 description 18
- TVYMKYUSZSVOAG-ZLUOBGJFSA-N Cys-Ala-Ala Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(O)=O TVYMKYUSZSVOAG-ZLUOBGJFSA-N 0.000 description 18
- CVOZXIPULQQFNY-ZLUOBGJFSA-N Cys-Ala-Cys Chemical compound C[C@H](NC(=O)[C@@H](N)CS)C(=O)N[C@@H](CS)C(O)=O CVOZXIPULQQFNY-ZLUOBGJFSA-N 0.000 description 18
- AEJSNWMRPXAKCW-WHFBIAKZSA-N Cys-Ala-Gly Chemical compound SC[C@H](N)C(=O)N[C@@H](C)C(=O)NCC(O)=O AEJSNWMRPXAKCW-WHFBIAKZSA-N 0.000 description 18
- 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 18
- 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 18
- GVVKYKCOFMMTKZ-WHFBIAKZSA-N Gly-Cys-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](CS)NC(=O)CN GVVKYKCOFMMTKZ-WHFBIAKZSA-N 0.000 description 18
- ZKJZBRHRWKLVSJ-ZDLURKLDSA-N Gly-Thr-Cys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)CN)O ZKJZBRHRWKLVSJ-ZDLURKLDSA-N 0.000 description 18
- JBVSSSZFNTXJDX-YTLHQDLWSA-N Ala-Ala-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@H](C)N JBVSSSZFNTXJDX-YTLHQDLWSA-N 0.000 description 15
- DECCMEWNXSNSDO-ZLUOBGJFSA-N Ala-Cys-Ala Chemical compound C[C@H](N)C(=O)N[C@@H](CS)C(=O)N[C@@H](C)C(O)=O DECCMEWNXSNSDO-ZLUOBGJFSA-N 0.000 description 15
- ZVFVBBGVOILKPO-WHFBIAKZSA-N Ala-Gly-Ala Chemical compound C[C@H](N)C(=O)NCC(=O)N[C@@H](C)C(O)=O ZVFVBBGVOILKPO-WHFBIAKZSA-N 0.000 description 15
- YRNBANYVJJBGDI-VZFHVOOUSA-N Thr-Ala-Cys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](C)C(=O)N[C@@H](CS)C(=O)O)N)O YRNBANYVJJBGDI-VZFHVOOUSA-N 0.000 description 15
- XPNSAQMEAVSQRD-FBCQKBJTSA-N Thr-Gly-Gly Chemical compound C[C@@H](O)[C@H](N)C(=O)NCC(=O)NCC(O)=O XPNSAQMEAVSQRD-FBCQKBJTSA-N 0.000 description 15
- UQJUGHFKNKGHFQ-VZFHVOOUSA-N Ala-Cys-Thr Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CS)C(=O)N[C@@H]([C@@H](C)O)C(O)=O UQJUGHFKNKGHFQ-VZFHVOOUSA-N 0.000 description 13
- HYKFOHGZGLOCAY-ZLUOBGJFSA-N Cys-Cys-Ala Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CS)C(=O)N[C@@H](C)C(O)=O HYKFOHGZGLOCAY-ZLUOBGJFSA-N 0.000 description 12
- 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 12
- QSDKBRMVXSWAQE-BFHQHQDPSA-N Gly-Ala-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)CN QSDKBRMVXSWAQE-BFHQHQDPSA-N 0.000 description 12
- JQFILXICXLDTRR-FBCQKBJTSA-N Gly-Thr-Gly Chemical compound NCC(=O)N[C@@H]([C@H](O)C)C(=O)NCC(O)=O JQFILXICXLDTRR-FBCQKBJTSA-N 0.000 description 12
- DGOJNGCGEYOBKN-BWBBJGPYSA-N Thr-Cys-Cys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CS)C(=O)O)N)O DGOJNGCGEYOBKN-BWBBJGPYSA-N 0.000 description 12
- SLUWOCTZVGMURC-BFHQHQDPSA-N Thr-Gly-Ala Chemical compound C[C@@H](O)[C@H](N)C(=O)NCC(=O)N[C@@H](C)C(O)=O SLUWOCTZVGMURC-BFHQHQDPSA-N 0.000 description 12
- WOJJIRYPFAZEPF-YFKPBYRVSA-N 2-[[(2s)-2-[[2-[(2-azaniumylacetyl)amino]acetyl]amino]propanoyl]amino]acetate Chemical compound OC(=O)CNC(=O)[C@H](C)NC(=O)CNC(=O)CN WOJJIRYPFAZEPF-YFKPBYRVSA-N 0.000 description 9
- RLMISHABBKUNFO-WHFBIAKZSA-N Ala-Ala-Gly Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)NCC(O)=O RLMISHABBKUNFO-WHFBIAKZSA-N 0.000 description 9
- RCQRKPUXJAGEEC-ZLUOBGJFSA-N Ala-Cys-Cys Chemical compound C[C@H](N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CS)C(O)=O RCQRKPUXJAGEEC-ZLUOBGJFSA-N 0.000 description 9
- QKHWNPQNOHEFST-VZFHVOOUSA-N Ala-Thr-Cys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](C)N)O QKHWNPQNOHEFST-VZFHVOOUSA-N 0.000 description 9
- PYTZFYUXZZHOAD-WHFBIAKZSA-N Gly-Ala-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)CN PYTZFYUXZZHOAD-WHFBIAKZSA-N 0.000 description 9
- 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 9
- 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 9
- FFJQHWKSGAWSTJ-BFHQHQDPSA-N Gly-Thr-Ala Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(O)=O FFJQHWKSGAWSTJ-BFHQHQDPSA-N 0.000 description 9
- WYKJENSCCRJLRC-ZDLURKLDSA-N Thr-Gly-Cys Chemical compound C[C@H]([C@@H](C(=O)NCC(=O)N[C@@H](CS)C(=O)O)N)O WYKJENSCCRJLRC-ZDLURKLDSA-N 0.000 description 9
- TZQWJCGVCIJDMU-HEIBUPTGSA-N Thr-Thr-Cys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CS)C(=O)O)N)O TZQWJCGVCIJDMU-HEIBUPTGSA-N 0.000 description 9
- 206010012601 diabetes mellitus Diseases 0.000 description 7
- 108010089804 glycyl-threonine Proteins 0.000 description 7
- QMOQBVOBWVNSNO-UHFFFAOYSA-N 2-[[2-[[2-[(2-azaniumylacetyl)amino]acetyl]amino]acetyl]amino]acetate Chemical compound NCC(=O)NCC(=O)NCC(=O)NCC(O)=O QMOQBVOBWVNSNO-UHFFFAOYSA-N 0.000 description 6
- WNHNMKOFKCHKKD-BFHQHQDPSA-N Ala-Thr-Gly Chemical compound [H]N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(O)=O WNHNMKOFKCHKKD-BFHQHQDPSA-N 0.000 description 6
- NRVQLLDIJJEIIZ-VZFHVOOUSA-N Cys-Thr-Ala Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](C)C(=O)O)NC(=O)[C@H](CS)N)O NRVQLLDIJJEIIZ-VZFHVOOUSA-N 0.000 description 6
- VNBNZUAPOYGRDB-ZDLURKLDSA-N Gly-Cys-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CS)NC(=O)CN)O VNBNZUAPOYGRDB-ZDLURKLDSA-N 0.000 description 6
- CCQOOWAONKGYKQ-BYPYZUCNSA-N Gly-Gly-Ala Chemical compound OC(=O)[C@H](C)NC(=O)CNC(=O)CN CCQOOWAONKGYKQ-BYPYZUCNSA-N 0.000 description 6
- IDOGEHIWMJMAHT-BYPYZUCNSA-N Gly-Gly-Cys Chemical compound NCC(=O)NCC(=O)N[C@@H](CS)C(O)=O IDOGEHIWMJMAHT-BYPYZUCNSA-N 0.000 description 6
- TVTZEOHWHUVYCG-KYNKHSRBSA-N Gly-Thr-Thr Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O TVTZEOHWHUVYCG-KYNKHSRBSA-N 0.000 description 6
- NXRNRBOKDBIVKQ-CXTHYWKRSA-N Ile-Tyr-Tyr Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CC2=CC=C(C=C2)O)C(=O)O)N NXRNRBOKDBIVKQ-CXTHYWKRSA-N 0.000 description 6
- PDIDTSZKKFEDMB-UWVGGRQHSA-N Lys-Pro-Gly Chemical compound [H]N[C@@H](CCCCN)C(=O)N1CCC[C@H]1C(=O)NCC(O)=O PDIDTSZKKFEDMB-UWVGGRQHSA-N 0.000 description 6
- IGROJMCBGRFRGI-YTLHQDLWSA-N Thr-Ala-Ala Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(O)=O IGROJMCBGRFRGI-YTLHQDLWSA-N 0.000 description 6
- 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 6
- NDZYTIMDOZMECO-SHGPDSBTSA-N Thr-Thr-Ala Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(O)=O NDZYTIMDOZMECO-SHGPDSBTSA-N 0.000 description 6
- 108010068265 aspartyltyrosine Proteins 0.000 description 6
- 210000004369 blood Anatomy 0.000 description 6
- 239000008280 blood Substances 0.000 description 6
- 108010016616 cysteinylglycine Proteins 0.000 description 6
- 108010084264 glycyl-glycyl-cysteine Proteins 0.000 description 6
- 108010067216 glycyl-glycyl-glycine Proteins 0.000 description 6
- 108010001064 glycyl-glycyl-glycyl-glycine Proteins 0.000 description 6
- 239000002773 nucleotide Substances 0.000 description 6
- 125000003729 nucleotide group Chemical group 0.000 description 6
- CLODWIOAKCSBAN-BQBZGAKWSA-N Gly-Arg-Asp Chemical compound NC(N)=NCCC[C@H](NC(=O)CN)C(=O)N[C@@H](CC(O)=O)C(O)=O CLODWIOAKCSBAN-BQBZGAKWSA-N 0.000 description 5
- 238000011160 research Methods 0.000 description 5
- 208000001072 type 2 diabetes mellitus Diseases 0.000 description 5
- SSONCJTVDRSLNK-UHFFFAOYSA-N 2-methylprop-2-enoic acid;hydrochloride Chemical compound Cl.CC(=C)C(O)=O SSONCJTVDRSLNK-UHFFFAOYSA-N 0.000 description 4
- 238000009007 Diagnostic Kit Methods 0.000 description 4
- OCJRHJZKGGSPRW-IUCAKERBSA-N Glu-Lys-Gly Chemical compound NCCCC[C@@H](C(=O)NCC(O)=O)NC(=O)[C@@H](N)CCC(O)=O OCJRHJZKGGSPRW-IUCAKERBSA-N 0.000 description 4
- UIGMAMGZOJVTDN-WHFBIAKZSA-N Ser-Gly-Ser Chemical compound OC[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O UIGMAMGZOJVTDN-WHFBIAKZSA-N 0.000 description 4
- 108010086434 alanyl-seryl-glycine Proteins 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 108010050848 glycylleucine Proteins 0.000 description 4
- 230000035945 sensitivity Effects 0.000 description 4
- 108010073969 valyllysine Proteins 0.000 description 4
- YYSWCHMLFJLLBJ-ZLUOBGJFSA-N Ala-Ala-Ser Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O YYSWCHMLFJLLBJ-ZLUOBGJFSA-N 0.000 description 3
- BTYTYHBSJKQBQA-GCJQMDKQSA-N Ala-Asp-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](C)N)O BTYTYHBSJKQBQA-GCJQMDKQSA-N 0.000 description 3
- HFBFSOAKPUZCCO-ZLUOBGJFSA-N Ala-Cys-Asn Chemical compound C[C@@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(=O)N)C(=O)O)N HFBFSOAKPUZCCO-ZLUOBGJFSA-N 0.000 description 3
- VWEWCZSUWOEEFM-WDSKDSINSA-N Ala-Gly-Ala-Gly Chemical compound C[C@H](N)C(=O)NCC(=O)N[C@@H](C)C(=O)NCC(O)=O VWEWCZSUWOEEFM-WDSKDSINSA-N 0.000 description 3
- LJFNNUBZSZCZFN-WHFBIAKZSA-N Ala-Gly-Cys Chemical compound N[C@@H](C)C(=O)NCC(=O)N[C@@H](CS)C(=O)O LJFNNUBZSZCZFN-WHFBIAKZSA-N 0.000 description 3
- HOVPGJUNRLMIOZ-CIUDSAMLSA-N Ala-Ser-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@H](C)N HOVPGJUNRLMIOZ-CIUDSAMLSA-N 0.000 description 3
- OEVCHROQUIVQFZ-YTLHQDLWSA-N Ala-Thr-Ala Chemical compound C[C@H](N)C(=O)N[C@@H]([C@H](O)C)C(=O)N[C@@H](C)C(O)=O OEVCHROQUIVQFZ-YTLHQDLWSA-N 0.000 description 3
- YMIYZAOBQDRCPP-UHFFFAOYSA-N Ala-Thr-Cys-Cys Chemical compound CC(N)C(=O)NC(C(O)C)C(=O)NC(CS)C(=O)NC(CS)C(O)=O YMIYZAOBQDRCPP-UHFFFAOYSA-N 0.000 description 3
- CREYEAPXISDKSB-FQPOAREZSA-N Ala-Thr-Tyr Chemical compound [H]N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O CREYEAPXISDKSB-FQPOAREZSA-N 0.000 description 3
- GCTANJIJJROSLH-GVARAGBVSA-N Ala-Tyr-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)NC(=O)[C@H](C)N GCTANJIJJROSLH-GVARAGBVSA-N 0.000 description 3
- NGYHSXDNNOFHNE-AVGNSLFASA-N Arg-Pro-Leu Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(C)C)C(O)=O NGYHSXDNNOFHNE-AVGNSLFASA-N 0.000 description 3
- ISJWBVIYRBAXEB-CIUDSAMLSA-N Arg-Ser-Glu Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(O)=O ISJWBVIYRBAXEB-CIUDSAMLSA-N 0.000 description 3
- QHUOOCKNNURZSL-IHRRRGAJSA-N Arg-Tyr-Ser Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CO)C(O)=O QHUOOCKNNURZSL-IHRRRGAJSA-N 0.000 description 3
- NLRJGXZWTKXRHP-DCAQKATOSA-N Asn-Leu-Arg Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O NLRJGXZWTKXRHP-DCAQKATOSA-N 0.000 description 3
- HDHZCEDPLTVHFZ-GUBZILKMSA-N Asn-Leu-Glu Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O HDHZCEDPLTVHFZ-GUBZILKMSA-N 0.000 description 3
- QNFRBNZGVVKBNJ-PEFMBERDSA-N Asp-Ile-Gln Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)O)NC(=O)[C@H](CC(=O)O)N QNFRBNZGVVKBNJ-PEFMBERDSA-N 0.000 description 3
- MNQMTYSEKZHIDF-GCJQMDKQSA-N Asp-Thr-Ala Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(O)=O MNQMTYSEKZHIDF-GCJQMDKQSA-N 0.000 description 3
- 208000024172 Cardiovascular disease Diseases 0.000 description 3
- SMYXEYRYCLIPIL-ZLUOBGJFSA-N Cys-Cys-Cys Chemical compound SC[C@H](N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CS)C(O)=O SMYXEYRYCLIPIL-ZLUOBGJFSA-N 0.000 description 3
- CVLIHKBUPSFRQP-WHFBIAKZSA-N Cys-Gly-Ala Chemical compound [H]N[C@@H](CS)C(=O)NCC(=O)N[C@@H](C)C(O)=O CVLIHKBUPSFRQP-WHFBIAKZSA-N 0.000 description 3
- DZLQXIFVQFTFJY-BYPYZUCNSA-N Cys-Gly-Gly Chemical compound SC[C@H](N)C(=O)NCC(=O)NCC(O)=O DZLQXIFVQFTFJY-BYPYZUCNSA-N 0.000 description 3
- OZHXXYOHPLLLMI-CIUDSAMLSA-N Cys-Lys-Ala Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(O)=O OZHXXYOHPLLLMI-CIUDSAMLSA-N 0.000 description 3
- NAPULYCVEVVFRB-HEIBUPTGSA-N Cys-Thr-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@@H](N)CS NAPULYCVEVVFRB-HEIBUPTGSA-N 0.000 description 3
- OYTPNWYZORARHL-XHNCKOQMSA-N Gln-Ala-Pro Chemical compound C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CCC(=O)N)N OYTPNWYZORARHL-XHNCKOQMSA-N 0.000 description 3
- OACQOWPRWGNKTP-AVGNSLFASA-N Gln-Tyr-Asp Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(O)=O)C(O)=O OACQOWPRWGNKTP-AVGNSLFASA-N 0.000 description 3
- IESFZVCAVACGPH-PEFMBERDSA-N Glu-Asp-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](N)CCC(O)=O IESFZVCAVACGPH-PEFMBERDSA-N 0.000 description 3
- ZYRXTRTUCAVNBQ-GVXVVHGQSA-N Glu-Val-Lys Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CCC(=O)O)N ZYRXTRTUCAVNBQ-GVXVVHGQSA-N 0.000 description 3
- GQGAFTPXAPKSCF-WHFBIAKZSA-N Gly-Ala-Cys Chemical compound NCC(=O)N[C@@H](C)C(=O)N[C@@H](CS)C(=O)O GQGAFTPXAPKSCF-WHFBIAKZSA-N 0.000 description 3
- UGVQELHRNUDMAA-BYPYZUCNSA-N Gly-Ala-Gly Chemical compound [NH3+]CC(=O)N[C@@H](C)C(=O)NCC([O-])=O UGVQELHRNUDMAA-BYPYZUCNSA-N 0.000 description 3
- GRIRDMVMJJDZKV-RCOVLWMOSA-N Gly-Asn-Val Chemical compound [H]NCC(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C(C)C)C(O)=O GRIRDMVMJJDZKV-RCOVLWMOSA-N 0.000 description 3
- CEXINUGNTZFNRY-BYPYZUCNSA-N Gly-Cys-Gly Chemical compound [NH3+]CC(=O)N[C@@H](CS)C(=O)NCC([O-])=O CEXINUGNTZFNRY-BYPYZUCNSA-N 0.000 description 3
- MIIVFRCYJABHTQ-ONGXEEELSA-N Gly-Leu-Val Chemical compound [H]NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(O)=O MIIVFRCYJABHTQ-ONGXEEELSA-N 0.000 description 3
- RVGMVLVBDRQVKB-UWVGGRQHSA-N Gly-Met-His Chemical compound CSCC[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)NC(=O)CN RVGMVLVBDRQVKB-UWVGGRQHSA-N 0.000 description 3
- WNZOCXUOGVYYBJ-CDMKHQONSA-N Gly-Phe-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)NC(=O)CN)O WNZOCXUOGVYYBJ-CDMKHQONSA-N 0.000 description 3
- XHVONGZZVUUORG-WEDXCCLWSA-N Gly-Thr-Lys Chemical compound NCC(=O)N[C@@H]([C@H](O)C)C(=O)N[C@H](C(O)=O)CCCCN XHVONGZZVUUORG-WEDXCCLWSA-N 0.000 description 3
- CSTDQOOBZBAJKE-BWAGICSOSA-N His-Tyr-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)NC(=O)[C@H](CC2=CN=CN2)N)O CSTDQOOBZBAJKE-BWAGICSOSA-N 0.000 description 3
- PJLLMGWWINYQPB-PEFMBERDSA-N Ile-Asn-Gln Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N PJLLMGWWINYQPB-PEFMBERDSA-N 0.000 description 3
- RWYCOSAAAJBJQL-KCTSRDHCSA-N Ile-Gly-Trp Chemical compound CC[C@H](C)[C@@H](C(=O)NCC(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)O)N RWYCOSAAAJBJQL-KCTSRDHCSA-N 0.000 description 3
- CAHCWMVNBZJVAW-NAKRPEOUSA-N Ile-Pro-Ser Chemical compound CC[C@H](C)[C@@H](C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(=O)O)N CAHCWMVNBZJVAW-NAKRPEOUSA-N 0.000 description 3
- PMGDADKJMCOXHX-UHFFFAOYSA-N L-Arginyl-L-glutamin-acetat Natural products NC(=N)NCCCC(N)C(=O)NC(CCC(N)=O)C(O)=O PMGDADKJMCOXHX-UHFFFAOYSA-N 0.000 description 3
- 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 3
- AXZGZMGRBDQTEY-SRVKXCTJSA-N Leu-Gln-Met Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCSC)C(O)=O AXZGZMGRBDQTEY-SRVKXCTJSA-N 0.000 description 3
- HPBCTWSUJOGJSH-MNXVOIDGSA-N Leu-Glu-Ile Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O HPBCTWSUJOGJSH-MNXVOIDGSA-N 0.000 description 3
- FEHQLKKBVJHSEC-SZMVWBNQSA-N Leu-Glu-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)CC(C)C)C(O)=O)=CNC2=C1 FEHQLKKBVJHSEC-SZMVWBNQSA-N 0.000 description 3
- MVVSHHJKJRZVNY-ACRUOGEOSA-N Leu-Phe-Tyr Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O MVVSHHJKJRZVNY-ACRUOGEOSA-N 0.000 description 3
- KIZIOFNVSOSKJI-CIUDSAMLSA-N Leu-Ser-Cys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CS)C(=O)O)N KIZIOFNVSOSKJI-CIUDSAMLSA-N 0.000 description 3
- AIMGJYMCTAABEN-GVXVVHGQSA-N Leu-Val-Glu Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O AIMGJYMCTAABEN-GVXVVHGQSA-N 0.000 description 3
- WZVSHTFTCYOFPL-GARJFASQSA-N Lys-Ser-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CO)NC(=O)[C@H](CCCCN)N)C(=O)O WZVSHTFTCYOFPL-GARJFASQSA-N 0.000 description 3
- SPSSJSICDYYTQN-HJGDQZAQSA-N Met-Thr-Gln Chemical compound CSCC[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@H](C(O)=O)CCC(N)=O SPSSJSICDYYTQN-HJGDQZAQSA-N 0.000 description 3
- 208000001145 Metabolic Syndrome Diseases 0.000 description 3
- CUMXHKAOHNWRFQ-BZSNNMDCSA-N Phe-Asp-Tyr Chemical compound C([C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)C1=CC=CC=C1 CUMXHKAOHNWRFQ-BZSNNMDCSA-N 0.000 description 3
- NAXPHWZXEXNDIW-JTQLQIEISA-N Phe-Gly-Gly Chemical compound OC(=O)CNC(=O)CNC(=O)[C@@H](N)CC1=CC=CC=C1 NAXPHWZXEXNDIW-JTQLQIEISA-N 0.000 description 3
- XDMMOISUAHXXFD-SRVKXCTJSA-N Phe-Ser-Asp Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(O)=O XDMMOISUAHXXFD-SRVKXCTJSA-N 0.000 description 3
- OYEDZGNMSBZCIM-XGEHTFHBSA-N Ser-Arg-Thr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(O)=O OYEDZGNMSBZCIM-XGEHTFHBSA-N 0.000 description 3
- ULVMNZOKDBHKKI-ACZMJKKPSA-N Ser-Gln-Asp Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O ULVMNZOKDBHKKI-ACZMJKKPSA-N 0.000 description 3
- YMTLKLXDFCSCNX-BYPYZUCNSA-N Ser-Gly-Gly Chemical compound OC[C@H](N)C(=O)NCC(=O)NCC(O)=O YMTLKLXDFCSCNX-BYPYZUCNSA-N 0.000 description 3
- SFTZWNJFZYOLBD-ZDLURKLDSA-N Ser-Gly-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CO SFTZWNJFZYOLBD-ZDLURKLDSA-N 0.000 description 3
- UIPXCLNLUUAMJU-JBDRJPRFSA-N Ser-Ile-Ser Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(O)=O UIPXCLNLUUAMJU-JBDRJPRFSA-N 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
- XUDRHBPSPAPDJP-SRVKXCTJSA-N Ser-Lys-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CO XUDRHBPSPAPDJP-SRVKXCTJSA-N 0.000 description 3
- AZWNCEBQZXELEZ-FXQIFTODSA-N Ser-Pro-Ser Chemical compound OC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(O)=O AZWNCEBQZXELEZ-FXQIFTODSA-N 0.000 description 3
- SRSPTFBENMJHMR-WHFBIAKZSA-N Ser-Ser-Gly Chemical compound OC[C@H](N)C(=O)N[C@@H](CO)C(=O)NCC(O)=O SRSPTFBENMJHMR-WHFBIAKZSA-N 0.000 description 3
- TYVAWPFQYFPSBR-BFHQHQDPSA-N Thr-Ala-Gly Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(=O)NCC(O)=O TYVAWPFQYFPSBR-BFHQHQDPSA-N 0.000 description 3
- GLQFKOVWXPPFTP-VEVYYDQMSA-N Thr-Arg-Asp Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(O)=O GLQFKOVWXPPFTP-VEVYYDQMSA-N 0.000 description 3
- LYGKYFKSZTUXGZ-ZDLURKLDSA-N Thr-Cys-Gly Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CS)C(=O)NCC(O)=O LYGKYFKSZTUXGZ-ZDLURKLDSA-N 0.000 description 3
- GXUWHVZYDAHFSV-FLBSBUHZSA-N Thr-Ile-Thr Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(O)=O GXUWHVZYDAHFSV-FLBSBUHZSA-N 0.000 description 3
- AHERARIZBPOMNU-KATARQTJSA-N Thr-Ser-Leu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(O)=O AHERARIZBPOMNU-KATARQTJSA-N 0.000 description 3
- UQCNIMDPYICBTR-KYNKHSRBSA-N Thr-Thr-Gly Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(O)=O UQCNIMDPYICBTR-KYNKHSRBSA-N 0.000 description 3
- 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 3
- MNYNCKZAEIAONY-XGEHTFHBSA-N Thr-Val-Ser Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(O)=O MNYNCKZAEIAONY-XGEHTFHBSA-N 0.000 description 3
- SVGAWGVHFIYAEE-JSGCOSHPSA-N Trp-Gly-Gln Chemical compound C1=CC=C2C(C[C@H](N)C(=O)NCC(=O)N[C@@H](CCC(N)=O)C(O)=O)=CNC2=C1 SVGAWGVHFIYAEE-JSGCOSHPSA-N 0.000 description 3
- MBLJBGZWLHTJBH-SZMVWBNQSA-N Trp-Val-Arg Chemical compound C1=CC=C2C(C[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O)=CNC2=C1 MBLJBGZWLHTJBH-SZMVWBNQSA-N 0.000 description 3
- YLRLHDFMMWDYTK-KKUMJFAQSA-N Tyr-Cys-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CS)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 YLRLHDFMMWDYTK-KKUMJFAQSA-N 0.000 description 3
- RIJPHPUJRLEOAK-JYJNAYRXSA-N Tyr-Gln-His Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CC2=CN=CN2)C(=O)O)N)O RIJPHPUJRLEOAK-JYJNAYRXSA-N 0.000 description 3
- HRHYJNLMIJWGLF-BZSNNMDCSA-N Tyr-Ser-Phe Chemical compound C([C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=C(O)C=C1 HRHYJNLMIJWGLF-BZSNNMDCSA-N 0.000 description 3
- 201000000690 abdominal obesity-metabolic syndrome Diseases 0.000 description 3
- 108010008355 arginyl-glutamine Proteins 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 108010063718 gamma-glutamylaspartic acid Proteins 0.000 description 3
- 108010078144 glutaminyl-glycine Proteins 0.000 description 3
- 108010015792 glycyllysine Proteins 0.000 description 3
- 238000013386 optimize process Methods 0.000 description 3
- 108010070409 phenylalanyl-glycyl-glycine Proteins 0.000 description 3
- 108010051242 phenylalanylserine Proteins 0.000 description 3
- 108010020755 prolyl-glycyl-glycine Proteins 0.000 description 3
- 108010029020 prolylglycine Proteins 0.000 description 3
- 239000000523 sample Substances 0.000 description 3
- 210000000952 spleen Anatomy 0.000 description 3
- 108010072986 threonyl-seryl-lysine Proteins 0.000 description 3
- 108010003137 tyrosyltyrosine Proteins 0.000 description 3
- 102100031251 1-acylglycerol-3-phosphate O-acyltransferase PNPLA3 Human genes 0.000 description 2
- 108050003337 1-acylglycerol-3-phosphate O-acyltransferase PNPLA3 Proteins 0.000 description 2
- RAQMSGVCGSJKCL-FOHZUACHSA-N Asn-Gly-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CC(N)=O RAQMSGVCGSJKCL-FOHZUACHSA-N 0.000 description 2
- OIMUAKUQOUEPCZ-WHFBIAKZSA-N Cys-Asn-Gly Chemical compound SC[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)NCC(O)=O OIMUAKUQOUEPCZ-WHFBIAKZSA-N 0.000 description 2
- DJTXYXZNNDDEOU-WHFBIAKZSA-N Gly-Asn-Cys Chemical compound C([C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)CN)C(=O)N DJTXYXZNNDDEOU-WHFBIAKZSA-N 0.000 description 2
- GGEJHJIXRBTJPD-BYPYZUCNSA-N Gly-Asn-Gly Chemical compound NCC(=O)N[C@@H](CC(N)=O)C(=O)NCC(O)=O GGEJHJIXRBTJPD-BYPYZUCNSA-N 0.000 description 2
- AFWYPMDMDYCKMD-KBPBESRZSA-N Gly-Leu-Tyr Chemical compound NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 AFWYPMDMDYCKMD-KBPBESRZSA-N 0.000 description 2
- 206010022489 Insulin Resistance Diseases 0.000 description 2
- KZNQNBZMBZJQJO-UHFFFAOYSA-N N-glycyl-L-proline Natural products NCC(=O)N1CCCC1C(O)=O KZNQNBZMBZJQJO-UHFFFAOYSA-N 0.000 description 2
- 108010002311 N-glycylglutamic acid Proteins 0.000 description 2
- WSRWHZRUOCACLJ-UWVGGRQHSA-N Pro-Gly-His Chemical compound C([C@@H](C(=O)O)NC(=O)CNC(=O)[C@H]1NCCC1)C1=CN=CN1 WSRWHZRUOCACLJ-UWVGGRQHSA-N 0.000 description 2
- ABSSTGUCBCDKMU-UWVGGRQHSA-N Pro-Lys-Gly Chemical compound NCCCC[C@@H](C(=O)NCC(O)=O)NC(=O)[C@@H]1CCCN1 ABSSTGUCBCDKMU-UWVGGRQHSA-N 0.000 description 2
- 210000001789 adipocyte Anatomy 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 108010047857 aspartylglycine Proteins 0.000 description 2
- 208000015606 cardiovascular system disease Diseases 0.000 description 2
- 208000029078 coronary artery disease Diseases 0.000 description 2
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical group NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 229940088597 hormone Drugs 0.000 description 2
- 239000005556 hormone Substances 0.000 description 2
- 238000003317 immunochromatography Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 229920001184 polypeptide Polymers 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 238000004879 turbidimetry Methods 0.000 description 2
- SCAKQYSGEIHPLV-IUCAKERBSA-N (4S)-4-[(2-aminoacetyl)amino]-5-[(2S)-2-(carboxymethylcarbamoyl)pyrrolidin-1-yl]-5-oxopentanoic acid Chemical compound NCC(=O)N[C@@H](CCC(O)=O)C(=O)N1CCC[C@H]1C(=O)NCC(O)=O SCAKQYSGEIHPLV-IUCAKERBSA-N 0.000 description 1
- CUVSTAMIHSSVKL-UWVGGRQHSA-N (4s)-4-[(2-aminoacetyl)amino]-5-[[(2s)-6-amino-1-(carboxymethylamino)-1-oxohexan-2-yl]amino]-5-oxopentanoic acid Chemical compound NCCCC[C@@H](C(=O)NCC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)CN CUVSTAMIHSSVKL-UWVGGRQHSA-N 0.000 description 1
- DQVAZKGVGKHQDS-UHFFFAOYSA-N 2-[[1-[2-[(2-amino-4-methylpentanoyl)amino]-4-methylpentanoyl]pyrrolidine-2-carbonyl]amino]-4-methylpentanoic acid Chemical compound CC(C)CC(N)C(=O)NC(CC(C)C)C(=O)N1CCCC1C(=O)NC(CC(C)C)C(O)=O DQVAZKGVGKHQDS-UHFFFAOYSA-N 0.000 description 1
- HVCOBJNICQPDBP-UHFFFAOYSA-N 3-[3-[3,5-dihydroxy-6-methyl-4-(3,4,5-trihydroxy-6-methyloxan-2-yl)oxyoxan-2-yl]oxydecanoyloxy]decanoic acid;hydrate Chemical compound O.OC1C(OC(CC(=O)OC(CCCCCCC)CC(O)=O)CCCCCCC)OC(C)C(O)C1OC1C(O)C(O)C(O)C(C)O1 HVCOBJNICQPDBP-UHFFFAOYSA-N 0.000 description 1
- LMFXXZPPZDCPTA-ZKWXMUAHSA-N Ala-Gly-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@H](C)N LMFXXZPPZDCPTA-ZKWXMUAHSA-N 0.000 description 1
- HAVKMRGWNXMCDR-STQMWFEESA-N Arg-Gly-Phe Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O HAVKMRGWNXMCDR-STQMWFEESA-N 0.000 description 1
- PRLPSDIHSRITSF-UNQGMJICSA-N Arg-Phe-Thr Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)O)C(O)=O PRLPSDIHSRITSF-UNQGMJICSA-N 0.000 description 1
- KXOPYFNQLVUOAQ-FXQIFTODSA-N Arg-Ser-Ala Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(O)=O KXOPYFNQLVUOAQ-FXQIFTODSA-N 0.000 description 1
- 206010003210 Arteriosclerosis Diseases 0.000 description 1
- VYLVOMUVLMGCRF-ZLUOBGJFSA-N Asn-Asp-Ser Chemical compound NC(=O)C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(O)=O VYLVOMUVLMGCRF-ZLUOBGJFSA-N 0.000 description 1
- LTZIRYMWOJHRCH-GUDRVLHUSA-N Asn-Ile-Pro Chemical compound CC[C@H](C)[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CC(=O)N)N LTZIRYMWOJHRCH-GUDRVLHUSA-N 0.000 description 1
- WCRQQIPFSXFIRN-LPEHRKFASA-N Asn-Met-Pro Chemical compound CSCC[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CC(=O)N)N WCRQQIPFSXFIRN-LPEHRKFASA-N 0.000 description 1
- QRULNKJGYQQZMW-ZLUOBGJFSA-N Asp-Asn-Asp Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O QRULNKJGYQQZMW-ZLUOBGJFSA-N 0.000 description 1
- NYQHSUGFEWDWPD-ACZMJKKPSA-N Asp-Gln-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CC(=O)O)N NYQHSUGFEWDWPD-ACZMJKKPSA-N 0.000 description 1
- SEMWSADZTMJELF-BYULHYEWSA-N Asp-Ile-Gly Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(O)=O SEMWSADZTMJELF-BYULHYEWSA-N 0.000 description 1
- LIVXPXUVXFRWNY-CIUDSAMLSA-N Asp-Lys-Ala Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(O)=O LIVXPXUVXFRWNY-CIUDSAMLSA-N 0.000 description 1
- HICVMZCGVFKTPM-BQBZGAKWSA-N Asp-Pro-Gly Chemical compound OC(=O)C[C@H](N)C(=O)N1CCC[C@H]1C(=O)NCC(O)=O HICVMZCGVFKTPM-BQBZGAKWSA-N 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
- AMRLSQGGERHDHJ-FXQIFTODSA-N Cys-Ala-Arg Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O AMRLSQGGERHDHJ-FXQIFTODSA-N 0.000 description 1
- JIVJXVJMOBVCJF-ZLUOBGJFSA-N Cys-Asn-Cys Chemical compound C([C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CS)N)C(=O)N JIVJXVJMOBVCJF-ZLUOBGJFSA-N 0.000 description 1
- VXDXZGYXHIADHF-YJRXYDGGSA-N Cys-Tyr-Thr Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H]([C@@H](C)O)C(O)=O VXDXZGYXHIADHF-YJRXYDGGSA-N 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 108700024394 Exon Proteins 0.000 description 1
- LLVXTGUTDYMJLY-GUBZILKMSA-N Gln-Asn-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CCC(=O)N)N LLVXTGUTDYMJLY-GUBZILKMSA-N 0.000 description 1
- CMBXOSFZCFGDLE-IHRRRGAJSA-N Gln-Tyr-Gln Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)O)NC(=O)[C@H](CCC(=O)N)N)O CMBXOSFZCFGDLE-IHRRRGAJSA-N 0.000 description 1
- CSMHMEATMDCQNY-DZKIICNBSA-N Gln-Val-Tyr Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O CSMHMEATMDCQNY-DZKIICNBSA-N 0.000 description 1
- CVPXINNKRTZBMO-CIUDSAMLSA-N Glu-Arg-Asn Chemical compound C(C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](CCC(=O)O)N)CN=C(N)N CVPXINNKRTZBMO-CIUDSAMLSA-N 0.000 description 1
- GLWXKFRTOHKGIT-ACZMJKKPSA-N Glu-Asn-Asn Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O GLWXKFRTOHKGIT-ACZMJKKPSA-N 0.000 description 1
- AIGROOHQXCACHL-WDSKDSINSA-N Glu-Gly-Ala Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H](C)C(O)=O AIGROOHQXCACHL-WDSKDSINSA-N 0.000 description 1
- OPAINBJQDQTGJY-JGVFFNPUSA-N Glu-Gly-Pro Chemical compound C1C[C@@H](N(C1)C(=O)CNC(=O)[C@H](CCC(=O)O)N)C(=O)O OPAINBJQDQTGJY-JGVFFNPUSA-N 0.000 description 1
- CAQXJMUDOLSBPF-SUSMZKCASA-N Glu-Thr-Thr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O CAQXJMUDOLSBPF-SUSMZKCASA-N 0.000 description 1
- PUUYVMYCMIWHFE-BQBZGAKWSA-N Gly-Ala-Arg Chemical compound NCC(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N PUUYVMYCMIWHFE-BQBZGAKWSA-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
- KFMBRBPXHVMDFN-UWVGGRQHSA-N Gly-Arg-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CCCNC(N)=N KFMBRBPXHVMDFN-UWVGGRQHSA-N 0.000 description 1
- XEJTYSCIXKYSHR-WDSKDSINSA-N Gly-Asp-Gln Chemical compound C(CC(=O)N)[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)CN XEJTYSCIXKYSHR-WDSKDSINSA-N 0.000 description 1
- XTQFHTHIAKKCTM-YFKPBYRVSA-N Gly-Glu-Gly Chemical compound NCC(=O)N[C@@H](CCC(O)=O)C(=O)NCC(O)=O XTQFHTHIAKKCTM-YFKPBYRVSA-N 0.000 description 1
- ULZCYBYDTUMHNF-IUCAKERBSA-N Gly-Leu-Glu Chemical compound NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O ULZCYBYDTUMHNF-IUCAKERBSA-N 0.000 description 1
- JPVGHHQGKPQYIL-KBPBESRZSA-N Gly-Phe-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CC1=CC=CC=C1 JPVGHHQGKPQYIL-KBPBESRZSA-N 0.000 description 1
- MYXNLWDWWOTERK-BHNWBGBOSA-N Gly-Thr-Pro Chemical compound C[C@H]([C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)CN)O MYXNLWDWWOTERK-BHNWBGBOSA-N 0.000 description 1
- RJVZMGQMJOQIAX-GJZGRUSLSA-N Gly-Trp-Met Chemical compound [H]NCC(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CCSC)C(O)=O RJVZMGQMJOQIAX-GJZGRUSLSA-N 0.000 description 1
- 229930186217 Glycolipid Natural products 0.000 description 1
- ZRSJXIKQXUGKRB-TUBUOCAGSA-N His-Ile-Thr Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(O)=O ZRSJXIKQXUGKRB-TUBUOCAGSA-N 0.000 description 1
- 101000775469 Homo sapiens Adiponectin Proteins 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 108091092195 Intron Proteins 0.000 description 1
- 241000880493 Leptailurus serval Species 0.000 description 1
- HVJVUYQWFYMGJS-GVXVVHGQSA-N Leu-Glu-Val Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O HVJVUYQWFYMGJS-GVXVVHGQSA-N 0.000 description 1
- QJXHMYMRGDOHRU-NHCYSSNCSA-N Leu-Ile-Gly Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(O)=O QJXHMYMRGDOHRU-NHCYSSNCSA-N 0.000 description 1
- KYIIALJHAOIAHF-KKUMJFAQSA-N Leu-Leu-His Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CC1=CN=CN1 KYIIALJHAOIAHF-KKUMJFAQSA-N 0.000 description 1
- PJWOOBTYQNNRBF-BZSNNMDCSA-N Leu-Phe-Lys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCCCN)C(=O)O)N PJWOOBTYQNNRBF-BZSNNMDCSA-N 0.000 description 1
- OZTZJMUZVAVJGY-BZSNNMDCSA-N Leu-Tyr-His Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CC2=CN=CN2)C(=O)O)N OZTZJMUZVAVJGY-BZSNNMDCSA-N 0.000 description 1
- GKFNXYMAMKJSKD-NHCYSSNCSA-N Lys-Asp-Val Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O GKFNXYMAMKJSKD-NHCYSSNCSA-N 0.000 description 1
- KEPWSUPUFAPBRF-DKIMLUQUSA-N Lys-Ile-Phe Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O KEPWSUPUFAPBRF-DKIMLUQUSA-N 0.000 description 1
- GILLQRYAWOMHED-DCAQKATOSA-N Lys-Val-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CCCCN GILLQRYAWOMHED-DCAQKATOSA-N 0.000 description 1
- CHDYFPCQVUOJEB-ULQDDVLXSA-N Met-Leu-Phe Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 CHDYFPCQVUOJEB-ULQDDVLXSA-N 0.000 description 1
- HQCSLJFGZYOXHW-KKUMJFAQSA-N Phe-His-Cys Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CC2=CN=CN2)C(=O)N[C@@H](CS)C(=O)O)N HQCSLJFGZYOXHW-KKUMJFAQSA-N 0.000 description 1
- MVIJMIZJPHQGEN-IHRRRGAJSA-N Phe-Ser-Val Chemical compound CC(C)[C@@H](C([O-])=O)NC(=O)[C@H](CO)NC(=O)[C@@H]([NH3+])CC1=CC=CC=C1 MVIJMIZJPHQGEN-IHRRRGAJSA-N 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- CLNJSLSHKJECME-BQBZGAKWSA-N Pro-Gly-Ala Chemical compound OC(=O)[C@H](C)NC(=O)CNC(=O)[C@@H]1CCCN1 CLNJSLSHKJECME-BQBZGAKWSA-N 0.000 description 1
- FEPSEIDIPBMIOS-QXEWZRGKSA-N Pro-Gly-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H]1CCCN1 FEPSEIDIPBMIOS-QXEWZRGKSA-N 0.000 description 1
- HAEGAELAYWSUNC-WPRPVWTQSA-N Pro-Gly-Val Chemical compound [H]N1CCC[C@H]1C(=O)NCC(=O)N[C@@H](C(C)C)C(O)=O HAEGAELAYWSUNC-WPRPVWTQSA-N 0.000 description 1
- UQTNIFUCMBFWEJ-IWGUZYHVSA-N Thr-Asn Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@H](C(O)=O)CC(N)=O UQTNIFUCMBFWEJ-IWGUZYHVSA-N 0.000 description 1
- UHBPFYOQQPFKQR-JHEQGTHGSA-N Thr-Gln-Gly Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(N)=O)C(=O)NCC(O)=O UHBPFYOQQPFKQR-JHEQGTHGSA-N 0.000 description 1
- MPUMPERGHHJGRP-WEDXCCLWSA-N Thr-Gly-Lys Chemical compound C[C@H]([C@@H](C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)O)N)O MPUMPERGHHJGRP-WEDXCCLWSA-N 0.000 description 1
- JKGGPMOUIAAJAA-YEPSODPASA-N Thr-Gly-Val Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](C(C)C)C(O)=O JKGGPMOUIAAJAA-YEPSODPASA-N 0.000 description 1
- YJCVECXVYHZOBK-KNZXXDILSA-N Thr-Ile-Pro Chemical compound CC[C@H](C)[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H]([C@@H](C)O)N YJCVECXVYHZOBK-KNZXXDILSA-N 0.000 description 1
- MXNAOGFNFNKUPD-JHYOHUSXSA-N Thr-Phe-Thr Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)O)C(O)=O MXNAOGFNFNKUPD-JHYOHUSXSA-N 0.000 description 1
- BZTSQFWJNJYZSX-JRQIVUDYSA-N Thr-Tyr-Asp Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(O)=O)C(O)=O BZTSQFWJNJYZSX-JRQIVUDYSA-N 0.000 description 1
- KVEWWQRTAVMOFT-KJEVXHAQSA-N Thr-Tyr-Val Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](C(C)C)C(O)=O KVEWWQRTAVMOFT-KJEVXHAQSA-N 0.000 description 1
- 101710120037 Toxin CcdB Proteins 0.000 description 1
- MBFJIHUHHCJBSN-AVGNSLFASA-N Tyr-Asn-Gln Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O MBFJIHUHHCJBSN-AVGNSLFASA-N 0.000 description 1
- JWHOIHCOHMZSAR-QWRGUYRKSA-N Tyr-Asp-Gly Chemical compound OC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 JWHOIHCOHMZSAR-QWRGUYRKSA-N 0.000 description 1
- WTTRJMAZPDHPGS-KKXDTOCCSA-N Tyr-Phe-Ala Chemical compound C[C@H](NC(=O)[C@H](Cc1ccccc1)NC(=O)[C@@H](N)Cc1ccc(O)cc1)C(O)=O WTTRJMAZPDHPGS-KKXDTOCCSA-N 0.000 description 1
- QFHRUCJIRVILCK-YJRXYDGGSA-N Tyr-Thr-Cys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)N)O QFHRUCJIRVILCK-YJRXYDGGSA-N 0.000 description 1
- YKBUNNNRNZZUID-UFYCRDLUSA-N Tyr-Val-Tyr Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O YKBUNNNRNZZUID-UFYCRDLUSA-N 0.000 description 1
- ZLMFVXMJFIWIRE-FHWLQOOXSA-N Val-Trp-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CC1=CNC2=CC=CC=C21)NC(=O)[C@H](C(C)C)N ZLMFVXMJFIWIRE-FHWLQOOXSA-N 0.000 description 1
- PDASTHRLDFOZMG-JYJNAYRXSA-N Val-Tyr-Met Chemical compound CSCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)C(C)C)CC1=CC=C(O)C=C1 PDASTHRLDFOZMG-JYJNAYRXSA-N 0.000 description 1
- 108010005233 alanylglutamic acid Proteins 0.000 description 1
- 230000006909 anti-apoptosis Effects 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 208000011775 arteriosclerosis disease Diseases 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 210000000748 cardiovascular system Anatomy 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- AGOYDEPGAOXOCK-KCBOHYOISA-N clarithromycin Chemical compound O([C@@H]1[C@@H](C)C(=O)O[C@@H]([C@@]([C@H](O)[C@@H](C)C(=O)[C@H](C)C[C@](C)([C@H](O[C@H]2[C@@H]([C@H](C[C@@H](C)O2)N(C)C)O)[C@H]1C)OC)(C)O)CC)[C@H]1C[C@@](C)(OC)[C@@H](O)[C@H](C)O1 AGOYDEPGAOXOCK-KCBOHYOISA-N 0.000 description 1
- 229960002626 clarithromycin Drugs 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 229940104302 cytosine Drugs 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 239000012470 diluted sample Substances 0.000 description 1
- FSXRLASFHBWESK-UHFFFAOYSA-N dipeptide phenylalanyl-tyrosine Natural products C=1C=C(O)C=CC=1CC(C(O)=O)NC(=O)C(N)CC1=CC=CC=C1 FSXRLASFHBWESK-UHFFFAOYSA-N 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000002526 effect on cardiovascular system Effects 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 108010008237 glutamyl-valyl-glycine Proteins 0.000 description 1
- JYPCXBJRLBHWME-UHFFFAOYSA-N glycyl-L-prolyl-L-arginine Natural products NCC(=O)N1CCCC1C(=O)NC(CCCN=C(N)N)C(O)=O JYPCXBJRLBHWME-UHFFFAOYSA-N 0.000 description 1
- 108010019832 glycyl-asparaginyl-glycine Proteins 0.000 description 1
- 108010050475 glycyl-leucyl-tyrosine Proteins 0.000 description 1
- 108010025801 glycyl-prolyl-arginine Proteins 0.000 description 1
- 108010077515 glycylproline Proteins 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 108010025306 histidylleucine Proteins 0.000 description 1
- 108010085325 histidylproline Proteins 0.000 description 1
- 102000057799 human ADIPOQ Human genes 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 108010053037 kyotorphin Proteins 0.000 description 1
- 108010057821 leucylproline Proteins 0.000 description 1
- 238000004020 luminiscence type Methods 0.000 description 1
- 108010054155 lysyllysine Proteins 0.000 description 1
- 108010038320 lysylphenylalanine Proteins 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 108700023046 methionyl-leucyl-phenylalanine Proteins 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 108010015796 prolylisoleucine Proteins 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 239000011534 wash buffer Substances 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/26—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against hormones ; against hormone releasing or inhibiting factors
-
- 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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- 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
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
- C07K2317/14—Specific host cells or culture conditions, e.g. components, pH or temperature
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Endocrinology (AREA)
- Pathology (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- General Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Organic Chemistry (AREA)
- Food Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
本发明涉及脂联素单克隆抗体及抗体对，所述脂联素单克隆抗体有三种，所述三种脂联素单克隆抗体分别为CSB‑DA120AmN①、CSB‑DA120AmN③和CSB‑DA120AmN④；所述抗体对为CSB‑DA120AmN③‑CSB‑DA120AmN④。本发明还提供了上述脂联素单克隆抗体及抗体对的制备方法，并保护其在在胶乳增强比浊、双抗夹心法的免疫检测产品中，或在制备检测脂联素的试剂、药剂或试剂盒中的应用。本发明通过基因重组，获得性能更为优异的脂联素单克隆抗体和抗体对，稳定、高效、批间差小，适于作为诊断试剂的抗体原料。
Description
技术领域
本发明属生物技术领域，具体涉及脂联素单克隆抗体、抗体对及其制备方法和用途。
背景技术
脂联素(Adiponectin/ADPN)是脂肪细胞分泌的一种内源性生物活性多肽或蛋白质。脂联素是一种胰岛素增敏激素(An Insulin-sensitizing Hormone)，能改善小鼠的胰岛素抗性(Insulin resistance)和动脉硬化症；对人体的研究发现，脂联素水平能预示II型糖尿病和冠心病的发展。
脂联素由APM1基因编码，该基因位于3号染色体长臂第27区域内，此区域亦存在2型糖尿病及代谢综合征的易感基因，全长17kb，包含2个内含子和3个外显子，其转录由5端胞嘧啶开始，多肽编码的区域起始于外显子2，终止于外显子3。
心血管系统疾病是威胁人类生命健康的首敌，彻底阐明其发病机理并寻求相应的治疗措施，是当今世界医学界亟待攻克的一项重大课题。近年来的研究证明，以糖尿病为突出临床特征的代谢综合征是心血管疾病的“原凶”。控制糖尿病及代谢综合征的药物正成为心血管系统疾病防治的第一战场。脂联素主要是由脂肪细胞合成和分泌的细胞因子，能够调节糖脂代谢、抗炎症、抗凋亡、抑制氧化/硝化反应，从而直接或者间接地保护心血管系统。肥胖、2型糖尿病患者血浆脂联素下降，其对心血管保护作用也减弱。
目前，脂联素已经开始用于II型糖尿病和冠心病的检测，然而现有的脂联素检测试剂灵敏度和精度都还不能满足临床诊断的需求。
发明内容
本发明针对现有技术中存在的不足，提供一种脂联素单克隆抗体、抗体对及其制备方法和用途，通过基因重组，获得性能更为优异的脂联素单克隆抗体和抗体对，稳定、高效、批间差小，适于作为诊断试剂的抗体原料。
为解决上述问题，本发明的技术方案如下：
本发明保护脂联素单克隆抗体，所述脂联素单克隆抗体有三种，所述三种脂联素单克隆抗体分别为CSB-DA120AmN①、CSB-DA120AmN③和CSB-DA120AmN④；
所述CSB-DA120AmN①轻链可变区部分核苷酸序列为SEQ ID NO.4和SEQ ID NO.5，所述CSB-DA120AmN①重链可变区部分核苷酸序列为SEQ ID NO.6和SEQ ID NO.7；
所述CSB-DA120AmN③轻链可变区部分核苷酸序列为SEQ ID NO.8和SEQ ID NO.9，所述CSB-DA120AmN③重链可变区部分核苷酸序列为SEQ ID NO.10和SEQ ID NO.11；
所述CSB-DA120AmN④轻链可变区部分核苷酸序列为SEQ ID NO.12和SEQ IDNO.13，所述CSB-DA120AmN④重链可变区部分核苷酸序列如SEQ ID NO.14和SEQ ID NO.15。
本发明还保护上述脂联素单克隆抗体的制备方法，包括以下步骤：
步骤1)，采用酵母表达系统表达脂联素重组蛋白，蛋白的序列为SEQ ID NO.1；
步骤2)，分离纯化步骤1)得到的脂联素重组蛋白；
步骤3)，将步骤2)得到的脂联素重组蛋白免疫宿主动物；
步骤4)，获取步骤3)得到的免疫宿主动物的脾脏细胞，并通过脾脏细胞制备融合细胞；
步骤5)，采用步骤4)制得的融合细胞制备脂联素单克隆抗体。
在上述技术方案的基础上，本发明还能够有如下改进。
进一步，所述步骤1)中，采用酵母表达系统表达，包括以下步骤：
步骤a，合成序列SEQ ID NO.1的基因序列以及对应的亚克隆引物，并连接至pPIC9K质粒，得到的连接产物转化到感受态E.coli TOP10，转化后将菌液涂布于含卡拉霉素和氨苄青霉素的LB固体培养基，将培养皿置于37℃培养箱培养12h以上，挑选单菌落PCR和双酶切验证正确后，进行测序验证，得到测序正确的重组子；
步骤b，将步骤a得到的三个重组子接种到含LB液体培养基，37℃培养8～16h，无菌条件下吸取菌液保种，剩余菌液接种于含卡拉霉素和氨苄青霉素的LB液体培养基，37℃培养12h以上，然后大提质粒；
步骤c，用Sac1酶对上述大提质粒进行线性化，37℃水浴过夜后回收线性化质粒，然后用Eppendor电转化仪分别电转化线性化质粒于GS115感受态中，涂布、转化后的菌液于RBD固体培养基，30℃培养4～5天；
步骤d，从RDB平板上挑选20株单菌落，PCR扩增目的基因APM1，琼脂糖凝胶电泳大小正确的条带鉴定为阳性，得到17个阳性菌落，挑取这些菌落接种于1.5ml YPG液体培养基的试管中，30℃，培养48h以上，吸取适量菌液与甘油均匀混合后冷冻保存备用，将得到7种阳性株接种于1.5ml YPG液体培养基，30℃，培养24h以上，然后加入1％甲醇诱导，培养24h以上，再加入1％甲醇诱导，继续培养24h以上，最后离心收集上清，TCA沉淀后，SDS-PAGE分析重组脂联素表达量；
步骤e，将产量最高的细胞株经前培养24h以上，按1％接种量接种至1L的YPG液体培养基中，30℃，培养24h以上，然后装入500ml Backman离心瓶，离心处理，用含1％甲醇的YP重悬菌体，倒入原三角瓶中，并加入含1％甲醇的YP和200ul消泡剂，30℃，培养72h以上，每隔24h加入10ml甲醇诱导，最后离心取上清。
进一步，所述步骤2)中，所述分离纯化，包括以下步骤：将最终得到的上清过Ni-NTA层析柱，然后用0～500mM NaCl洗脱，收集A280吸收峰的蛋白，最后SDS-PAGE分析蛋白纯度，纯化蛋白浓缩后备用。
优选的，所述引物序列为SEQ ID NO.2和SEQ ID NO.3。
进一步，所述步骤3)中，所述宿主动物为Balb/C雌性小鼠，所述融合用细胞系SP2/0。
优选的，所述步骤3)中，所述脂联素重组蛋白免疫宿主动物包括以下步骤：以每只老鼠50ug脂联素重组蛋白加等量的弗氏完全佐剂，制成油包水乳剂，免疫6-8周龄Balb/C雌性小鼠；每两周免疫一次所述小鼠，每次免疫取50ug脂联素重组蛋白与弗氏不完全佐剂混匀；免疫四次后测效价，待效价达到1:10000以上时，按50ug/mouse腹腔冲击免疫。
进一步，所述步骤4)中，所述获取免疫宿主动物的脾脏细胞采用以下步骤，腹腔冲击免疫三天后，在无菌条件下取出小鼠脾脏，并制成单个脾细胞悬液。
进一步，所述步骤4)中，所述制备融合细胞工序，包括细胞融合和细胞建株，最后得到杂交瘤细胞。
优选的，所述步骤4)中，所述制备融合细胞工序包括以下步骤：
步骤A，细胞融合：制备脾细胞悬液并用PBS洗涤干净后混入SP2/0细胞，脾细胞和SP2/0以10:1的比例混合并离心，离心后滴干混合细胞，轻敲弹松细胞团块，在37℃水浴中加入1ml PEG1450，加完后在37℃水浴中反应2min，沿管壁缓慢加入20ml RPMI-1640终止液，融合细胞终止后于800rpm离心5min，并吸干残留液体，HAT培养基重悬融合细胞，转入96孔细胞培养板培养，待融合细胞在HAT中培养7天左右后换成HT培养基；
步骤B，细胞建株：待孔中融合版细胞长至中等大小约104个细胞以上时开始检测，分别取50ul细胞培养液加入预先包被脂联素蛋白的微孔中，检测细胞上清抗体效价，从检测为阳性对应的微孔取阳性细胞进行亚克隆，利用HT培养基通过有限稀释法将筛选得到细胞稀释至1个/孔，将细胞铺于96孔细胞培养板，待单克隆细胞长至中等大小，密度约104个细胞以上即可检测效价，然后再次选取其中阳性细胞，重复一次亚克隆筛选，待检测到所有微孔中细胞上清都是阳性后，再进行一次同样的单克隆，直至有限稀释法后再次得到全阳性结果，能确定此时筛选到阳性细胞株，所得的阳性细胞株即为杂交瘤细胞。
进一步，所述步骤5)中，融合细胞制备抗体，包括无血清制备和纯化步骤，纯化后得到不同的单克隆抗体。
优选的，所述步骤5)中，无血清制备包括以下步骤：在有血清条件下复苏杂交瘤细胞，在连续几次细胞传代过程中，梯度减少血清的比例，逐渐增加代用的添加物，使杂交瘤细胞适应并继续生长；用无血清培养基复苏杂交瘤细胞，在细胞状态良好时，加大培养基的用量及培养细胞的容器，待细胞大量死亡时，收夜，并离心取上清，过滤收集，取待纯化的样品上样Protein A-琼脂糖亲和层析柱，流速为0.5ml/min，使抗体与Protein A结合，最后用洗脱液进行洗脱，得到抗体SDS-PAGE并鉴定其纯度。
本发明还保护一种脂联素单克隆抗体对，所述脂联素单克隆抗体对为CSB-DA120AmN③-CSB-DA120AmN④。
本发明还保护上述脂联素单克隆抗体对的制备方法，包括如下步骤：
将所述的单克隆抗体CSB-DA120AmN③、CSB-DA120AmN④，分别用作捕获抗体和标记抗体，组合成抗体对用于双抗夹心法的免疫检测，将抗体对对各种浓度的脂联素重组蛋白进行检测，然后将单克隆抗体对应的细胞株提取RNA，经RT-PCR反转录得到cDNA，利用小鼠IgG抗体轻链和重链可变区通用引物进行DNA测序，确定抗体的可变区序列。
此外，本发明还保护上述脂联素单克隆抗体和/或脂联素单克隆抗体对，在胶乳增强比浊、双抗夹心法的免疫检测产品中，或在制备检测脂联素的试剂、药剂或试剂盒中的应用。
本发明的有益效果是：本发明采用酵母表达系统表达脂联素重组蛋白，并免疫小鼠，得到脾脏细胞，然后用脾脏细胞制备融合细胞即为杂交瘤细胞，杂交瘤细胞用无血清驯化、培养，上清过滤后纯化得到不同的单克隆抗体，进行效价验证之后取优选的抗体，后对单克隆抗体进行DNA测序，确定脂联素抗体可变区序列，本发明所述制备方法稳定，得到产物纯度程度高，批间差小；本发明的脂联素单克隆抗体、抗体对，具有稳定、高效等诸多优良特点，适用于作为诊断试剂盒开发的抗体原料，应用范围也较广，尤其适用于胶乳增强比浊、化学发光、免疫层析等高端诊断试剂盒开发的抗体原料；本发明的脂联素单克隆抗体、抗体对灵敏度较高，对各种浓度的脂联素重组蛋白进行定量检测，均能够满足临床检验的要求。
附图说明
图1为本发明实施例1抗体对11例健康志愿者血样和21例糖尿病患者血样在胶乳比浊平台进行检测的检测结果图；
图2为本发明实施例2抗体对14例健康志愿者血样和25例糖尿病患者血样在化学发光平台进行检测的检测结果图；
图3为本发明实施例2抗体对14例健康志愿者血样和25例糖尿病患者血样在免疫层析平台进行检测的检测结果图。
具体实施方式
以下对本发明的原理和特征进行描述，所举实例只用于解释本发明，并非用于限定本发明的范围。
本发明设计了脂联素单克隆抗体，所述脂联素单克隆抗体有三种，所述三种脂联素单克隆抗体分别为CSB-DA120AmN①、CSB-DA120AmN③和CSB-DA120AmN④；
所述CSB-DA120AmN①轻链可变区部分核苷酸序列为SEQ ID NO.4和SEQ ID NO.5，所述CSB-DA120AmN①重链可变区部分核苷酸序列为SEQ ID NO.6和SEQ ID NO.7；
所述CSB-DA120AmN③轻链可变区部分核苷酸序列为SEQ ID NO.8和SEQ ID NO.9，所述CSB-DA120AmN③重链可变区部分核苷酸序列为SEQ ID NO.10和SEQ ID NO.11；
所述CSB-DA120AmN④轻链可变区部分核苷酸序列为SEQ ID NO.12和SEQ IDNO.13，所述CSB-DA120AmN④重链可变区部分核苷酸序列如SEQ ID NO.14和SEQ ID NO.15。
目前，定量检测脂联素的检测试剂普遍采用脂联素单克隆抗体。如公开号为CN111239421A，发明名称为一种定量检测脂联素ADPN的胶乳增强免疫比浊试剂盒及其制备与应用方法的中国专利，比如公开号为CN104694478B，发明名称为抗人脂联素的单克隆抗体及其应用的中国专利，再比如公开号为CN110372783A，发明名称为一种脂联素抗原、抗体及其胶乳试剂的制备方法以及脂联素检测试剂盒的中国专利均采用了脂联素单克隆抗体，上述抗体均是依赖脂联素制备的，而且在制备过程中并未对脂联素的基因序列进行改造。
发明人通过对上述现有脂联素检测产品进行实验研究，发现上述产品脂联素的单克隆抗体的应用范围有限，而且检测灵敏度也存在一定问题。
针对上述问题，发明人对脂联素的基因进行了相关研究，并设计了脂联素单克隆抗体的基因重组方案，以克服上述问题。在研究过程中，发明人发现采用双抗夹心法免疫检测，效果较佳，并基于此发现设计了脂联素单克隆抗体对，以确保能够实现双抗夹心法免疫检测。
本发明所设计的脂联素单克隆抗体对为CSB-DA120AmN③-CSB-DA120AmN④。
采用抗体对后，抗体对的两个抗体分别用作捕获抗体和标记抗体，能够提供极高的检测灵敏度，可以对脂联素进行精确的定量检测。
上述脂联素单克隆抗体的制备方法，包括以下步骤：
步骤1)，采用酵母表达系统表达脂联素重组蛋白，蛋白的序列为SEQ ID NO.1；
步骤2)，分离纯化步骤1)得到的脂联素重组蛋白；
步骤3)，将步骤2)得到的脂联素重组蛋白免疫宿主动物；
步骤4)，获取步骤3)得到的免疫宿主动物的脾脏细胞，并通过脾脏细胞制备融合细胞；
步骤5)，采用步骤4)制得的融合细胞制备脂联素单克隆抗体。
本发明采用了酵母表达系统表达脂联素重组蛋白，可以成功实现胞内表达或是分泌表达，且其放大培养基相对廉价，培养条件要求不高，适宜工业放大，所得重组蛋白与天然蛋白十分接近。
同时，本发明通过脾脏细胞制备融合细胞，能够实现无血清驯化、培养，制备方法稳定，得到产物纯度程度高，批间差小。
具体的，所述步骤1)中，采用酵母表达系统表达，可以采用以下优化的工艺步骤：
步骤a，合成序列SEQ ID NO.1的基因序列以及对应的亚克隆引物，并连接至pPIC9K质粒，得到的连接产物转化到感受态E.coli TOP10，转化后将菌液涂布于含卡拉霉素和氨苄青霉素的LB固体培养基，将培养皿置于37℃培养箱培养12h以上，挑选单菌落PCR和双酶切验证正确后，进行测序验证，得到测序正确的重组子；
步骤b，将步骤a得到的三个重组子接种到含LB液体培养基，37℃培养8～16h，无菌条件下吸取菌液保种，剩余菌液接种于含卡拉霉素和氨苄青霉素的LB液体培养基，37℃培养12h以上，然后大提质粒；
步骤c，用Sac1酶对上述大提质粒进行线性化，37℃水浴过夜后回收线性化质粒，然后用Eppendor电转化仪分别电转化线性化质粒于GS115感受态中，涂布、转化后的菌液于RBD固体培养基，30℃培养4～5天；
步骤d，从RDB平板上挑选20株单菌落，PCR扩增目的基因APM1，琼脂糖凝胶电泳大小正确的条带鉴定为阳性，得到17个阳性菌落，挑取这些菌落接种于1.5ml YPG液体培养基的试管中，30℃，培养48h以上，吸取适量菌液与甘油均匀混合后冷冻保存备用，将得到7种阳性株接种于1.5ml YPG液体培养基，30℃，培养24h以上，然后加入1％甲醇诱导，培养24h以上，再加入1％甲醇诱导，继续培养24h以上，最后离心收集上清，TCA沉淀后，SDS-PAGE分析重组脂联素表达量；
步骤e，将产量最高的细胞株经前培养24h以上，按1％接种量接种至1L的YPG液体培养基中，30℃，培养24h以上，然后装入500ml Backman离心瓶，离心处理，用含1％甲醇的YP重悬菌体，倒入原三角瓶中，并加入含1％甲醇的YP和200ul消泡剂，30℃，培养72h以上，每隔24h加入10ml甲醇诱导，最后离心取上清。
在本发明较为优选的实施方式中，所述步骤2)中，所述分离纯化，包括以下步骤：将最终得到的上清过Ni-NTA层析柱，然后用0～500mM NaCl洗脱，收集A280吸收峰的蛋白，最后SDS-PAGE分析蛋白纯度，纯化蛋白浓缩后备用。
在本发明较为优选的实施方式中，所述引物序列为SEQ ID NO.2和SEQ ID NO.3。
在本发明较为优选的实施方式中，所述步骤3)中，所述宿主动物为Balb/C雌性小鼠，所述融合用细胞系SP2/0。
在本发明更较为优选的实施方式中，所述步骤3)中，所述脂联素重组蛋白免疫宿主动物包括以下步骤：以每只老鼠50ug脂联素重组蛋白加等量的弗氏完全佐剂，制成油包水乳剂，免疫6-8周龄Balb/C雌性小鼠；每两周免疫一次所述小鼠，每次免疫取50ug脂联素重组蛋白与弗氏不完全佐剂混匀；免疫四次后测效价，待效价达到1:10000以上时，按50ug/mouse腹腔冲击免疫。
选择采用Balb/C雌性小鼠作为宿主动物，融合用细胞系SP2/0，宿主动物个体差异小，遗传基因更纯，整体素质更好，融合产生杂交瘤细胞分裂能力更强，使单克隆抗体迅速大量增殖，有效提高制备效率和制备的稳定性。
在本发明较为优选的实施方式中，所述步骤4)中，所述获取免疫宿主动物的脾脏细胞采用以下步骤，腹腔冲击免疫三天后，在无菌条件下取出小鼠脾脏，并制成单个脾细胞悬液。
在本发明较为优选的实施方式中，所述步骤4)中，所述制备融合细胞工序，包括细胞融合和细胞建株，最后得到杂交瘤细胞。
具体的，所述制备融合细胞工序可以采用以下优化的工艺步骤：
步骤A，细胞融合：制备脾细胞悬液并用PBS洗涤干净后混入SP2/0细胞，脾细胞和SP2/0以10:1的比例混合并离心，离心后滴干混合细胞，轻敲弹松细胞团块，在37℃水浴中加入1ml PEG1450，加完后在37℃水浴中反应2min，沿管壁缓慢加入20ml RPMI-1640终止液，融合细胞终止后于800rpm离心5min，并吸干残留液体，HAT培养基重悬融合细胞，转入96孔细胞培养板培养，待融合细胞在HAT中培养7天左右后换成HT培养基；
步骤B，细胞建株：待孔中融合版细胞长至中等大小约104个细胞以上时开始检测，分别取50ul细胞培养液加入预先包被脂联素蛋白的微孔中，检测细胞上清抗体效价，从检测为阳性对应的微孔取阳性细胞进行亚克隆，利用HT培养基通过有限稀释法将筛选得到细胞稀释至1个/孔，将细胞铺于96孔细胞培养板，待单克隆细胞长至中等大小，密度约104个细胞以上即可检测效价，然后再次选取其中阳性细胞，重复一次亚克隆筛选，待检测到所有微孔中细胞上清都是阳性后，再进行一次同样的单克隆，直至有限稀释法后再次得到全阳性结果，能确定此时筛选到阳性细胞株，所得的阳性细胞株即为杂交瘤细胞。
在本发明较为优选的实施方式中，所述步骤5)中，融合细胞制备抗体，包括无血清制备和纯化步骤，纯化后得到不同的单克隆抗体。
具体的，所述无血清制备可以采用以下优化的工艺步骤：
在有血清条件下复苏杂交瘤细胞，在连续几次细胞传代过程中，梯度减少血清的比例，逐渐增加代用的添加物，使杂交瘤细胞适应并继续生长；用无血清培养基复苏杂交瘤细胞，在细胞状态良好时，加大培养基的用量及培养细胞的容器，待细胞大量死亡时，收夜，并离心取上清，过滤收集，取待纯化的样品上样Protein A-琼脂糖亲和层析柱，流速为0.5ml/min，使抗体与Protein A结合，最后用洗脱液进行洗脱，得到抗体SDS-PAGE并鉴定其纯度。
上述脂联素单克隆抗体对的制备方法，包括如下步骤：
将所述的单克隆抗体CSB-DA120AmN③、CSB-DA120AmN④，分别用作捕获抗体和标记抗体，组合成抗体对用于双抗夹心法的免疫检测，将抗体对对各种浓度的脂联素重组蛋白进行检测，然后将单克隆抗体对应的细胞株提取RNA，经RT-PCR反转录得到cDNA，利用小鼠IgG抗体轻链和重链可变区通用引物进行DNA测序，确定抗体的可变区序列。
此外，本发明还保护上述脂联素单克隆抗体和/或脂联素单克隆抗体对，在胶乳增强比浊、双抗夹心法的免疫检测产品中，或在制备检测脂联素的试剂、药剂或试剂盒中的应用。
实施例1
脂联素重组蛋白制备以及分离纯化；
A、合成序列表1的基因序列以及相应亚克隆引物(引物序列如SEQ ID NO.2和SEQID NO.3所述)并连接至pPIC9K质粒，得到的连接产物转化到感受态E.coli TOP10，转化后将菌液涂布于含50ug/ml卡拉霉素和1mg/ml氨苄青霉素的LB固体培养基，将培养皿置于37℃培养箱培养12h，挑选单菌落PCR和双酶切验证正确后，进行测序，得到测序正确的重组子；
B、将上述正确的三个重组子接种到含3ml LB液体培养基，200rpm，37℃培养8-16h，无菌条件下吸取200ul菌液保种，剩下的2.8ml菌液接种于300ml LB液体培养基(含50ug/ml卡拉霉素和1mg/ml氨苄青霉素)，200rpm，37℃培养12h，然后大提质粒，质粒纯度99％以上，浓度在10ug/ul左右；
C、用Sac1酶对上述大提质粒进行线性化，37℃水浴过夜后回收线性化质粒，然后用Eppendor电转化仪分别电转化(电压1500V，时间5ms)线性化质粒于GS115感受态中；涂布150ul转化后的菌液于(1mg/ml G418)RBD固体培养基，30℃培养4-5天；
D、小量表达筛选：从RDB平板上挑选20株单菌落，PCR扩增目的基因APM1，琼脂糖凝胶电泳大小正确的条带鉴定为阳性，得到17个阳性菌落，挑取这些菌落接种于1.5ml YPG液体培养基的试管中，30℃，200rpm培养48h，吸取一定量菌液与甘油均匀混合后保存于-20℃备用，将得到7种阳性株接种于1.5ml YPG液体培养基，30℃，200rpm培养24h，然后加入1％甲醇诱导，培养24h再加入1％甲醇诱导，继续培养24h后离心收集上清，TCA沉淀后，SDS-PAGE分析重组脂联素表达量，最高产量约3mg/ml。
E、摇瓶发酵：将产量最高的细胞株经前培养24h，按1％接种量接种至1L的YPG液体培养基中，30℃，200rpm培养24h，然后装入500ml Backman离心瓶，8000rpm，3min离心，用YP(含1％甲醇)重悬菌体，倒入原来的三角瓶中，并加入YP(含1％甲醇)和200ul消泡剂，30℃，200rpm培养72h，每隔24h加入10ml甲醇诱导；最后离心取上清，浓缩后纯化。
F、将上清过Ni-NTA层析柱，然后用0-500mM NaCl(PH8.0)洗脱，收集A280吸收峰的蛋白，最后SDS-PAGE分析蛋白纯度，纯化蛋白浓缩后备用。
实施例2
脂联素单克隆抗体制备
2.1脂联素重组蛋白免疫小鼠
按每只老鼠50ug脂联素重组蛋白，加等量的弗氏完全佐剂，制成油包水乳剂，免疫6-8周龄Balb/C雌性小鼠；每两周免疫一次，每次免疫取50ug酵母表达重组蛋白与弗氏不完全佐剂混匀。免疫四次后测效价，若效价达到1:10000以上，按50ug/mouse腹腔冲击免疫，三天后在无菌条件下取出小鼠脾脏，并制成单个细胞悬液。
2.2细胞融合
制备脾细胞悬液并用PBS洗涤干净后混入SP2/0细胞，按脾细胞：SP2/0＝10:1混合并离心，1000rpm离心5min后滴干混合细胞，轻敲弹松细胞团快。在37℃水浴中加入1mlPEG1450，加完后再37℃水浴中反应2min，沿管壁缓慢加入20ml RPMI—1640终止液。
融合细胞终止后于800rpm离心5min，并吸干残留液体。HAT培养基重悬融合细胞，转入96孔细胞培养板培养，待融合细胞在HAT中培养7天左右后换成HT培养基。
2.3细胞建株
待孔中融合版细胞长至中等大小约104个细胞以上时开始检测，分别取50ul细胞培养液加入预先包被脂联素蛋白的微孔中，检测细胞上清抗体效价(即阴性对照<1.0，阳性对照>1.0)，从检测为阳性对应的微孔中取阳性细胞进行亚克隆。利用HT培养基通过有限稀释法，将筛选得到细胞稀释至1个/孔，将细胞铺于96孔细胞培养板，待克隆细胞长至中等大小，密度约104个细胞以上即可检测效价，然后再次取其中阳性细胞重复一次亚克隆筛选，待检测到所有微孔中细胞上清都是阳性后，再进行一次同样单克隆，如果有限稀释法后再次得到全阳性结果，能确定此时筛选到阳性细胞株，所得到的的阳性细胞株即为杂交瘤细胞。
2.4无血清培养杂交瘤细胞及纯化抗体
在有血清条件下复苏杂交瘤细胞，在连续几次细胞传代过程中，梯度减少血清的比例(如20％血清降至15％血清降至10％血清依次到无血清)，逐渐增加代用的添加物，使杂交瘤细胞能够很快适应并继续生长。在制备单克隆抗体时，用无血清培养基复苏杂交瘤细胞，在细胞状态良好时，加大培养基的用量及培养细胞的容器，待细胞大量死亡时，收夜，并离心取上清，过滤收集，取待纯化的样品上样Protein A-琼脂糖亲和层析柱(HiTrapProtein G,Pharmacia Biotech)流速为0.5ml/min，使抗体与Protein A结合，最后用洗脱液进行洗脱，得到抗体SDS-PAGE鉴定其纯度。
2.5抗体验证
CSB-DA120AmN①；
CSB-DA120AmN③—CSB-DA120AmN④
脂联素单克隆抗体对的制备方法，包括如下步骤：将所述的单克隆抗体CSB-DA120AmN③、CSB-DA120AmN④，分别用作捕获抗体和标记抗体，组合成抗体对应用于双抗夹心法的免疫检测，将抗体对对各种浓度的脂联素重组蛋白进行检测，然后将单克隆抗体对应的细胞株提取RNA，经RT-PCR反转录得到cDNA，利用小鼠IgG抗体轻链和重链可变区通用引物进行DNA测序，确定抗体的可变区序列。
CSB-DA120AmN①、CSB-DA120AmN③和CSB-DA120AmN④：
CSB-DA120AmN①轻链可变区部分核苷酸序列如SEQ ID NO.4和SEQ ID NO.5所述：
CSB-DA120AmN①重链可变区部分核苷酸序列如SEQ ID NO.6和SEQ ID NO.7所述：
CSB-DA120AmN③轻链可变区部分核苷酸序列如SEQ ID NO.8和SEQ ID NO.9所述：
CSB-DA120AmN③重链可变区部分核苷酸序列如SEQ ID NO.10和SEQ ID NO.11所述：
CSB-DA120AmN④轻链可变区部分核苷酸序列如SEQ ID NO.12和SEQ ID NO.13所述；
CSB-DA120AmN④重链可变区部分核苷酸序列如SEQ ID NO.14和SEQ ID NO.15所述。
本发明实施例制备的抗体临床应用效果评价如下：
为验证本发明实施例制备的抗体对适用于临床检测，利用抗体对组合对14例健康志愿者血样和25例糖尿病患者血样进行检测，其检测结果如附图2所示，此结果表明本发明实施例所产生的抗体能区分正常组和病例组样本，能够满足临床检测需求。
检测的实验流程如下：
A、用0.05M PH9.0碳酸盐包被缓冲液将抗体稀释至浓度为1ug/ml，后在每个聚苯乙烯板的反应孔中加0.1ml抗体溶液，4℃过夜。次日，弃去孔内溶液，用洗涤缓冲液洗3次，每次3分钟；
B、加稀释后的待检样本50ul于上述已包被的反应孔中，置37℃孵育1小时，然后洗涤；
C、于个反应孔中，加入稀释的酶标抗体100ul，37℃孵育1小时，然后洗涤；
D、加入化学发光底物液50ul，避光放置于微孔板化学发光仪上，静置5min，依序测量各孔的相对发光单位(RLU)。
由图1～图3的数据结果可知，采用本发明实施例的方法制备的脂联素单克隆抗体，具有稳定、高效等诸多优良特点，适用于作为诊断试剂盒开发的抗体原料，尤其适用于胶乳增强比浊、化学发光、免疫层析等高端诊断试剂盒开发的抗体原料。
以上所述仅为本发明的较佳实施例，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
序列表
<110> 武汉华美生物工程有限公司
<120> 脂联素单克隆抗体、抗体对及其制备方法和用途
<160> 15
<170> SIPOSequenceListing 1.0
<210> 1
<211> 226
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Glu Thr Thr Thr Gln Gly Pro Gly Val Leu Leu Pro Leu Pro Lys Gly
1 5 10 15
Ala Cys Thr Gly Trp Met Ala Gly Ile Pro Gly His Pro Gly His Asn
20 25 30
Gly Ala Pro Gly Arg Asp Gly Arg Asp Gly Thr Pro Gly Glu Lys Gly
35 40 45
Glu Lys Gly Asp Pro Gly Leu Ile Gly Pro Lys Gly Asp Ile Gly Glu
50 55 60
Thr Gly Val Pro Gly Ala Glu Gly Pro Arg Gly Phe Pro Gly Ile Gln
65 70 75 80
Gly Arg Lys Gly Glu Pro Gly Glu Gly Ala Tyr Val Tyr Arg Ser Ala
85 90 95
Phe Ser Val Gly Leu Glu Thr Tyr Val Thr Ile Pro Asn Met Pro Ile
100 105 110
Arg Phe Thr Lys Ile Phe Tyr Asn Gln Gln Asn His Tyr Asp Gly Ser
115 120 125
Thr Gly Lys Phe His Cys Asn Ile Pro Gly Leu Tyr Tyr Phe Ala Tyr
130 135 140
His Ile Thr Val Tyr Met Lys Asp Val Lys Val Ser Leu Phe Lys Lys
145 150 155 160
Asp Lys Ala Met Leu Phe Thr Tyr Asp Gln Tyr Gln Glu Asn Asn Val
165 170 175
Asp Gln Ala Ser Gly Ser Val Leu Leu His Leu Glu Val Gly Asp Gln
180 185 190
Val Trp Leu Gln Val Tyr Gly Glu Gly Glu Arg Asn Gly Leu Tyr Ala
195 200 205
Asp Asn Asp Asn Asp Ser Thr Phe Thr Gly Phe Leu Leu Tyr His Asp
210 215 220
Thr Asn
225
<210> 2
<211> 25
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Gly Ala Arg Ala Cys Asn Ala Cys Asn Ala Cys Asn Cys Ala Arg Gly
1 5 10 15
Gly Asn Cys Cys Asn Gly Gly Asn Gly
20 25
<210> 3
<211> 25
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
Cys Asn Cys Cys Asn Gly Gly Asn Cys Cys Tyr Thr Gly Asn Gly Thr
1 5 10 15
Asn Gly Thr Asn Gly Thr Tyr Thr Cys
20 25
<210> 4
<211> 321
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Gly Ala Thr Ala Thr Cys Cys Ala Gly Ala Thr Gly Ala Cys Thr Cys
1 5 10 15
Ala Gly Thr Cys Thr Cys Cys Ala Thr Cys Cys Thr Cys Ala Cys Thr
20 25 30
Gly Thr Cys Thr Gly Cys Ala Thr Cys Thr Cys Thr Gly Gly Gly Ala
35 40 45
Gly Gly Cys Ala Ala Thr Gly Thr Cys Ala Cys Cys Ala Thr Cys Ala
50 55 60
Cys Thr Thr Gly Cys Ala Ala Gly Gly Cys Ala Ala Gly Cys Cys Ala
65 70 75 80
Ala Gly Ala Cys Ala Thr Thr Ala Ala Cys Cys Ala Gly Thr Ala Thr
85 90 95
Ala Thr Ala Gly Gly Thr Thr Gly Gly Thr Ala Cys Cys Ala Ala Cys
100 105 110
Ala Cys Ala Ala Gly Cys Cys Thr Gly Gly Ala Ala Ala Ala Ala Gly
115 120 125
Thr Cys Cys Thr Ala Gly Gly Cys Cys Gly Cys Thr Cys Ala Thr Ala
130 135 140
Cys Ala Thr Thr Ala Cys Ala Cys Ala Thr Cys Thr Ala Ala Ala Thr
145 150 155 160
Thr Ala Cys Thr Gly Gly Cys Ala Gly Gly Cys Ala Thr Cys Cys Cys
165 170 175
Ala Thr Cys Ala Ala Gly Gly Thr Ala Cys Ala Gly Thr Gly Gly Ala
180 185 190
Ala Gly Thr Gly Gly Gly Thr Cys Thr Gly Gly Gly Ala Gly Ala Gly
195 200 205
Ala Thr Thr Ala Thr Thr Cys Cys Thr Thr Cys Ala Gly Cys Ala Thr
210 215 220
Cys Ala Gly Cys Ala Ala Cys Cys Thr Gly Gly Ala Gly Cys Cys Thr
225 230 235 240
Gly Ala Ala Gly Ala Thr Ala Thr Thr Gly Cys Gly Ala Cys Thr Thr
245 250 255
Ala Thr Thr Ala Thr Thr Gly Thr Cys Thr Ala Cys Ala Ala Thr Ala
260 265 270
Thr Gly Ala Thr Ala Ala Thr Cys Thr Thr Cys Gly Gly Ala Cys Gly
275 280 285
Thr Thr Cys Gly Gly Thr Gly Gly Ala Gly Gly Cys Ala Cys Ala Ala
290 295 300
Ala Gly Thr Thr Gly Gly Ala Ala Ala Thr Ala Ala Ala Ala Cys Gly
305 310 315 320
Gly
<210> 5
<211> 107
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 5
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Gly Asn Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Gln Tyr
20 25 30
Ile Gly Trp Tyr Gln His Lys Pro Gly Lys Ser Pro Arg Pro Leu Ile
35 40 45
His Tyr Thr Ser Lys Leu Leu Ala Gly Ile Pro Ser Arg Tyr Ser Gly
50 55 60
Ser Gly Ser Gly Arg Asp Tyr Ser Phe Ser Ile Ser Asn Leu Glu Pro
65 70 75 80
Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Asn Leu Arg Thr
85 90 95
Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
100 105
<210> 6
<211> 348
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 6
Gly Ala Gly Gly Thr Gly Ala Ala Gly Cys Thr Gly Gly Thr Gly Gly
1 5 10 15
Ala Ala Thr Cys Thr Gly Gly Gly Gly Gly Ala Gly Gly Cys Thr Thr
20 25 30
Ala Gly Thr Gly Ala Ala Gly Cys Cys Thr Gly Gly Ala Gly Gly Gly
35 40 45
Thr Cys Cys Cys Gly Gly Ala Cys Ala Cys Thr Cys Thr Cys Cys Thr
50 55 60
Gly Thr Gly Cys Ala Gly Cys Cys Thr Cys Thr Gly Gly Ala Thr Thr
65 70 75 80
Cys Ala Cys Thr Thr Thr Cys Ala Gly Thr Gly Ala Cys Thr Ala Thr
85 90 95
Gly Gly Ala Ala Thr Gly Cys Ala Cys Thr Gly Gly Gly Thr Cys Cys
100 105 110
Gly Thr Cys Ala Gly Gly Cys Thr Cys Cys Ala Gly Ala Gly Ala Ala
115 120 125
Gly Gly Gly Gly Cys Thr Gly Gly Ala Gly Thr Gly Gly Ala Thr Thr
130 135 140
Gly Cys Ala Thr Ala Cys Ala Thr Thr Ala Gly Thr Ala Gly Thr Gly
145 150 155 160
Gly Cys Ala Gly Thr Gly Gly Thr Ala Cys Cys Ala Thr Cys Thr Ala
165 170 175
Cys Thr Ala Thr Gly Cys Ala Gly Ala Cys Ala Cys Ala Thr Thr Gly
180 185 190
Ala Ala Gly Gly Gly Cys Cys Gly Ala Thr Thr Cys Ala Cys Cys Ala
195 200 205
Thr Cys Thr Cys Cys Ala Gly Ala Gly Ala Cys Ala Ala Thr Gly Cys
210 215 220
Cys Ala Thr Gly Ala Ala Ala Ala Thr Cys Cys Thr Gly Thr Thr Cys
225 230 235 240
Cys Thr Gly Cys Ala Ala Ala Thr Gly Ala Cys Cys Ala Gly Thr Cys
245 250 255
Thr Gly Ala Gly Gly Thr Cys Thr Gly Ala Gly Gly Ala Cys Ala Cys
260 265 270
Ala Gly Cys Cys Ala Thr Thr Thr Ala Thr Thr Ala Cys Thr Gly Thr
275 280 285
Ala Cys Ala Ala Gly Gly Gly Ala Thr Cys Thr Gly Thr Thr Cys Thr
290 295 300
Ala Cys Thr Thr Thr Gly Ala Cys Thr Ala Cys Thr Gly Gly Gly Gly
305 310 315 320
Cys Cys Ala Ala Gly Gly Cys Ala Cys Cys Ala Cys Thr Cys Thr Cys
325 330 335
Ala Cys Ala Gly Thr Cys Thr Cys Cys Thr Cys Ala
340 345
<210> 7
<211> 116
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 7
Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly
1 5 10 15
Ser Arg Thr Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr
20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Ile
35 40 45
Ala Tyr Ile Ser Ser Gly Ser Gly Thr Ile Tyr Tyr Ala Asp Thr Leu
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Met Lys Ile Leu Phe
65 70 75 80
Leu Gln Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Ile Tyr Tyr Cys
85 90 95
Thr Arg Asp Leu Phe Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu
100 105 110
Thr Val Ser Ser
115
<210> 8
<211> 321
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 8
Gly Ala Thr Ala Thr Cys Cys Ala Gly Ala Thr Gly Ala Cys Thr Cys
1 5 10 15
Ala Gly Thr Cys Thr Cys Cys Ala Thr Cys Cys Thr Cys Ala Cys Thr
20 25 30
Gly Thr Cys Thr Gly Cys Ala Thr Cys Thr Cys Thr Gly Gly Gly Ala
35 40 45
Gly Gly Cys Ala Ala Thr Gly Thr Cys Ala Cys Cys Ala Thr Cys Ala
50 55 60
Cys Thr Thr Gly Cys Ala Ala Gly Gly Cys Ala Ala Gly Cys Cys Ala
65 70 75 80
Ala Gly Ala Cys Ala Thr Thr Ala Ala Cys Cys Ala Gly Thr Ala Thr
85 90 95
Ala Thr Ala Gly Gly Thr Thr Gly Gly Thr Ala Cys Cys Ala Ala Cys
100 105 110
Ala Cys Ala Ala Gly Cys Cys Thr Gly Gly Ala Ala Ala Ala Ala Gly
115 120 125
Thr Cys Cys Thr Ala Gly Gly Cys Cys Gly Cys Thr Cys Ala Thr Ala
130 135 140
Cys Ala Thr Thr Ala Cys Ala Cys Ala Thr Cys Thr Ala Ala Ala Thr
145 150 155 160
Thr Ala Cys Thr Gly Gly Cys Ala Gly Gly Cys Ala Thr Cys Cys Cys
165 170 175
Ala Thr Cys Ala Ala Gly Gly Thr Ala Cys Ala Gly Thr Gly Gly Ala
180 185 190
Ala Gly Thr Gly Gly Gly Thr Cys Thr Gly Gly Gly Ala Gly Ala Gly
195 200 205
Ala Thr Thr Ala Thr Thr Cys Cys Thr Thr Cys Ala Gly Cys Ala Thr
210 215 220
Cys Ala Gly Cys Ala Ala Cys Cys Thr Gly Gly Ala Gly Cys Cys Thr
225 230 235 240
Gly Ala Ala Gly Ala Thr Ala Thr Thr Gly Cys Gly Ala Cys Thr Thr
245 250 255
Ala Thr Thr Ala Thr Thr Gly Thr Cys Thr Ala Cys Ala Ala Thr Ala
260 265 270
Thr Gly Ala Thr Ala Ala Thr Cys Thr Thr Cys Gly Gly Ala Cys Gly
275 280 285
Thr Thr Cys Gly Gly Thr Gly Gly Ala Gly Gly Cys Ala Cys Ala Ala
290 295 300
Ala Gly Thr Thr Gly Gly Ala Ala Ala Thr Ala Ala Ala Ala Cys Gly
305 310 315 320
Gly
<210> 9
<211> 107
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 9
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Gly Asn Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Gln Tyr
20 25 30
Ile Gly Trp Tyr Gln His Lys Pro Gly Lys Ser Pro Arg Pro Leu Ile
35 40 45
His Tyr Thr Ser Lys Leu Leu Ala Gly Ile Pro Ser Arg Tyr Ser Gly
50 55 60
Ser Gly Ser Gly Arg Asp Tyr Ser Phe Ser Ile Ser Asn Leu Glu Pro
65 70 75 80
Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Asn Leu Arg Thr
85 90 95
Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
100 105
<210> 10
<211> 348
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 10
Gly Ala Gly Gly Thr Gly Ala Ala Gly Cys Thr Gly Gly Thr Gly Gly
1 5 10 15
Ala Ala Thr Cys Thr Gly Gly Gly Gly Gly Ala Gly Gly Cys Thr Thr
20 25 30
Ala Gly Thr Gly Ala Ala Gly Cys Cys Thr Gly Gly Ala Gly Gly Gly
35 40 45
Thr Cys Cys Cys Gly Gly Ala Cys Ala Cys Thr Cys Thr Cys Cys Thr
50 55 60
Gly Thr Gly Cys Ala Gly Cys Cys Thr Cys Thr Gly Gly Ala Thr Thr
65 70 75 80
Cys Ala Cys Thr Thr Thr Cys Ala Gly Thr Gly Ala Cys Thr Ala Thr
85 90 95
Gly Gly Ala Ala Thr Gly Cys Ala Cys Thr Gly Gly Gly Thr Cys Cys
100 105 110
Gly Thr Cys Ala Gly Gly Cys Thr Cys Cys Ala Gly Ala Gly Ala Ala
115 120 125
Gly Gly Gly Gly Cys Thr Gly Gly Ala Gly Thr Gly Gly Ala Thr Thr
130 135 140
Gly Cys Ala Thr Ala Cys Ala Thr Thr Ala Gly Thr Ala Gly Thr Gly
145 150 155 160
Gly Cys Ala Gly Thr Gly Gly Thr Ala Cys Cys Ala Thr Cys Thr Ala
165 170 175
Cys Thr Ala Thr Gly Cys Ala Gly Ala Cys Ala Cys Ala Thr Thr Gly
180 185 190
Ala Ala Gly Gly Gly Cys Cys Gly Ala Thr Thr Cys Ala Cys Cys Ala
195 200 205
Thr Cys Thr Cys Cys Ala Gly Ala Gly Ala Cys Ala Ala Thr Gly Cys
210 215 220
Cys Ala Thr Gly Ala Ala Ala Ala Thr Cys Cys Thr Gly Thr Thr Cys
225 230 235 240
Cys Thr Gly Cys Ala Ala Ala Thr Gly Ala Cys Cys Ala Gly Thr Cys
245 250 255
Thr Gly Ala Gly Gly Thr Cys Thr Gly Ala Gly Gly Ala Cys Ala Cys
260 265 270
Ala Gly Cys Cys Ala Thr Thr Thr Ala Thr Thr Ala Cys Thr Gly Thr
275 280 285
Ala Cys Ala Ala Gly Gly Gly Ala Thr Cys Thr Gly Thr Thr Cys Thr
290 295 300
Ala Cys Thr Thr Thr Gly Ala Cys Thr Ala Cys Thr Gly Gly Gly Gly
305 310 315 320
Cys Cys Ala Ala Gly Gly Cys Ala Cys Cys Ala Cys Thr Cys Thr Cys
325 330 335
Ala Cys Ala Gly Thr Cys Thr Cys Cys Thr Cys Ala
340 345
<210> 11
<211> 116
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 11
Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly
1 5 10 15
Ser Arg Thr Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr
20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Ile
35 40 45
Ala Tyr Ile Ser Ser Gly Ser Gly Thr Ile Tyr Tyr Ala Asp Thr Leu
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Met Lys Ile Leu Phe
65 70 75 80
Leu Gln Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Ile Tyr Tyr Cys
85 90 95
Thr Arg Asp Leu Phe Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu
100 105 110
Thr Val Ser Ser
115
<210> 12
<211> 321
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 12
Gly Ala Thr Ala Thr Cys Cys Ala Gly Ala Thr Gly Ala Cys Thr Cys
1 5 10 15
Ala Gly Thr Cys Thr Cys Cys Ala Thr Cys Cys Thr Cys Ala Cys Thr
20 25 30
Gly Thr Cys Thr Gly Cys Ala Thr Cys Thr Cys Thr Gly Gly Gly Ala
35 40 45
Gly Gly Cys Ala Ala Thr Gly Thr Cys Ala Cys Cys Ala Thr Cys Ala
50 55 60
Cys Thr Thr Gly Cys Ala Ala Gly Gly Cys Ala Ala Gly Cys Cys Ala
65 70 75 80
Ala Gly Ala Cys Ala Thr Thr Ala Ala Cys Cys Ala Gly Thr Ala Thr
85 90 95
Ala Thr Ala Gly Gly Thr Thr Gly Gly Thr Ala Cys Cys Ala Ala Cys
100 105 110
Ala Cys Ala Ala Gly Cys Cys Thr Gly Gly Ala Ala Ala Ala Ala Gly
115 120 125
Thr Cys Cys Thr Ala Gly Gly Cys Cys Gly Cys Thr Cys Ala Thr Ala
130 135 140
Cys Ala Thr Thr Ala Cys Ala Cys Ala Thr Cys Thr Ala Ala Ala Thr
145 150 155 160
Thr Ala Cys Thr Gly Gly Cys Ala Gly Gly Cys Ala Thr Cys Cys Cys
165 170 175
Ala Thr Cys Ala Ala Gly Gly Thr Ala Cys Ala Gly Thr Gly Gly Ala
180 185 190
Ala Gly Thr Gly Gly Gly Thr Cys Thr Gly Gly Gly Ala Gly Ala Gly
195 200 205
Ala Thr Thr Ala Thr Thr Cys Cys Thr Thr Cys Ala Gly Cys Ala Thr
210 215 220
Cys Ala Gly Cys Ala Ala Cys Cys Thr Gly Gly Ala Gly Cys Cys Thr
225 230 235 240
Gly Ala Ala Gly Ala Thr Ala Thr Thr Gly Cys Gly Ala Cys Thr Thr
245 250 255
Ala Thr Thr Ala Thr Thr Gly Thr Cys Thr Ala Cys Ala Ala Thr Ala
260 265 270
Thr Gly Ala Thr Ala Ala Thr Cys Thr Thr Cys Gly Gly Ala Cys Gly
275 280 285
Thr Thr Cys Gly Gly Thr Gly Gly Ala Gly Gly Cys Ala Cys Ala Ala
290 295 300
Ala Gly Thr Thr Gly Gly Ala Ala Ala Thr Ala Ala Ala Ala Cys Gly
305 310 315 320
Gly
<210> 13
<211> 107
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 13
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Gly Asn Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Gln Tyr
20 25 30
Ile Gly Trp Tyr Gln His Lys Pro Gly Lys Ser Pro Arg Pro Leu Ile
35 40 45
His Tyr Thr Ser Lys Leu Leu Ala Gly Ile Pro Ser Arg Tyr Ser Gly
50 55 60
Ser Gly Ser Gly Arg Asp Tyr Ser Phe Ser Ile Ser Asn Leu Glu Pro
65 70 75 80
Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Asn Leu Arg Thr
85 90 95
Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
100 105
<210> 14
<211> 348
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 14
Gly Ala Gly Gly Thr Gly Ala Ala Gly Cys Thr Gly Gly Thr Gly Gly
1 5 10 15
Ala Ala Thr Cys Thr Gly Gly Gly Gly Gly Ala Gly Gly Cys Thr Thr
20 25 30
Ala Gly Thr Gly Ala Ala Gly Cys Cys Thr Gly Gly Ala Gly Gly Gly
35 40 45
Thr Cys Cys Cys Gly Gly Ala Cys Ala Cys Thr Cys Thr Cys Cys Thr
50 55 60
Gly Thr Gly Cys Ala Gly Cys Cys Thr Cys Thr Gly Gly Ala Thr Thr
65 70 75 80
Cys Ala Cys Thr Thr Thr Cys Ala Gly Thr Gly Ala Cys Thr Ala Thr
85 90 95
Gly Gly Ala Ala Thr Gly Cys Ala Cys Thr Gly Gly Gly Thr Cys Cys
100 105 110
Gly Thr Cys Ala Gly Gly Cys Thr Cys Cys Ala Gly Ala Gly Ala Ala
115 120 125
Gly Gly Gly Gly Cys Thr Gly Gly Ala Gly Thr Gly Gly Ala Thr Thr
130 135 140
Gly Cys Ala Thr Ala Cys Ala Thr Thr Ala Gly Thr Ala Gly Thr Gly
145 150 155 160
Gly Cys Ala Gly Thr Gly Gly Thr Ala Cys Cys Ala Thr Cys Thr Ala
165 170 175
Cys Thr Ala Thr Gly Cys Ala Gly Ala Cys Ala Cys Ala Thr Thr Gly
180 185 190
Ala Ala Gly Gly Gly Cys Cys Gly Ala Thr Thr Cys Ala Cys Cys Ala
195 200 205
Thr Cys Thr Cys Cys Ala Gly Ala Gly Ala Cys Ala Ala Thr Gly Cys
210 215 220
Cys Ala Thr Gly Ala Ala Ala Ala Thr Cys Cys Thr Gly Thr Thr Cys
225 230 235 240
Cys Thr Gly Cys Ala Ala Ala Thr Gly Ala Cys Cys Ala Gly Thr Cys
245 250 255
Thr Gly Ala Gly Gly Thr Cys Thr Gly Ala Gly Gly Ala Cys Ala Cys
260 265 270
Ala Gly Cys Cys Ala Thr Thr Thr Ala Thr Thr Ala Cys Thr Gly Thr
275 280 285
Ala Cys Ala Ala Gly Gly Gly Ala Thr Cys Thr Gly Thr Thr Cys Thr
290 295 300
Ala Cys Thr Thr Thr Gly Ala Cys Thr Ala Cys Thr Gly Gly Gly Gly
305 310 315 320
Cys Cys Ala Ala Gly Gly Cys Ala Cys Cys Ala Cys Thr Cys Thr Cys
325 330 335
Ala Cys Ala Gly Thr Cys Thr Cys Cys Thr Cys Ala
340 345
<210> 15
<211> 116
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 15
Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly
1 5 10 15
Ser Arg Thr Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr
20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Ile
35 40 45
Ala Tyr Ile Ser Ser Gly Ser Gly Thr Ile Tyr Tyr Ala Asp Thr Leu
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Met Lys Ile Leu Phe
65 70 75 80
Leu Gln Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Ile Tyr Tyr Cys
85 90 95
Thr Arg Asp Leu Phe Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu
100 105 110
Thr Val Ser Ser
115
Claims (15)
1.脂联素单克隆抗体，其特征在于：所述脂联素单克隆抗体有三种，所述三种脂联素单克隆抗体分别为CSB-DA120AmN①、CSB-DA120AmN③和CSB-DA120AmN④；
所述CSB-DA120AmN①轻链可变区部分核苷酸序列为SEQ ID NO.4和SEQ ID NO.5，所述CSB-DA120AmN①重链可变区部分核苷酸序列为SEQ ID NO.6和SEQ ID NO.7；
所述CSB-DA120AmN③轻链可变区部分核苷酸序列为SEQ ID NO.8和SEQ ID NO.9，所述CSB-DA120AmN③重链可变区部分核苷酸序列为SEQ ID NO.10和SEQ ID NO.11；
所述CSB-DA120AmN④轻链可变区部分核苷酸序列为SEQ ID NO.12和SEQ ID NO.13，所述CSB-DA120AmN④重链可变区部分核苷酸序列如SEQ ID NO.14和SEQ ID NO.15。
2.如权利要求1所述脂联素单克隆抗体的制备方法，其特征在于包括以下步骤：
步骤1)，采用酵母表达系统表达脂联素重组蛋白，蛋白的序列为SEQ ID NO.1；
步骤2)，分离纯化步骤1)得到的脂联素重组蛋白；
步骤3)，将步骤2)得到的脂联素重组蛋白免疫宿主动物；
步骤4)，获取步骤3)得到的免疫宿主动物的脾脏细胞，并通过脾脏细胞制备融合细胞；
步骤5)，采用步骤4)制得的融合细胞制备脂联素单克隆抗体。
3.根据权利要求2所述的脂联素单克隆抗体的制备方法，其特征在于，所述步骤1)中，采用酵母表达系统表达，包括以下步骤：
步骤a，合成序列SEQ ID NO.1的基因序列以及对应的亚克隆引物，并连接至pPIC9K质粒，得到的连接产物转化到感受态E.coli TOP10，转化后将菌液涂布于含卡拉霉素和氨苄青霉素的LB固体培养基，将培养皿置于37℃培养箱培养12h以上，挑选单菌落PCR和双酶切验证正确后，进行测序验证，得到测序正确的重组子；
步骤b，将步骤a得到的三个重组子接种到含LB液体培养基，37℃培养8～16h，无菌条件下吸取菌液保种，剩余菌液接种于含卡拉霉素和氨苄青霉素的LB液体培养基，37℃培养12h以上，然后大提质粒；
步骤c，用Sac1酶对上述大提质粒进行线性化，37℃水浴过夜后回收线性化质粒，然后用Eppendor电转化仪分别电转化线性化质粒于GS115感受态中，涂布、转化后的菌液于RBD固体培养基，30℃培养4～5天；
步骤d，从RDB平板上挑选20株单菌落，PCR扩增目的基因APM1，琼脂糖凝胶电泳大小正确的条带鉴定为阳性，得到17个阳性菌落，挑取这些菌落接种于1.5mlYPG液体培养基的试管中，30℃，培养48h以上，吸取适量菌液与甘油均匀混合后冷冻保存备用，将得到7种阳性株接种于1.5mlYPG液体培养基，30℃，培养24h以上，然后加入1％甲醇诱导，培养24h以上，再加入1％甲醇诱导，继续培养24h以上，最后离心收集上清，TCA沉淀后，SDS-PAGE分析重组脂联素表达量；
步骤e，将产量最高的细胞株经前培养24h以上，按1％接种量接种至1L的YPG液体培养基中，30℃，培养24h以上，然后装入500ml Backman离心瓶，离心处理，用含1％甲醇的YP重悬菌体，倒入原三角瓶中，并加入含1％甲醇的YP和200ul消泡剂，30℃，培养72h以上，每隔24h加入10ml甲醇诱导，最后离心取上清。
4.根据权利要求3所述的脂联素单克隆抗体的制备方法，其特征在于，所述步骤2)中，所述分离纯化，包括以下步骤：将最终得到的上清过Ni-NTA层析柱，然后用0～500mM NaCl洗脱，收集A280吸收峰的蛋白，最后SDS-PAGE分析蛋白纯度，纯化蛋白浓缩后备用。
5.根据权利要求3所述的脂联素单克隆抗体的制备方法，其特征在于，所述引物序列为SEQ ID NO.2和SEQ ID NO.3。
6.根据权利要求2所述的脂联素单克隆抗体的制备方法，其特征在于，所述步骤3)中，所述宿主动物为Balb/C雌性小鼠，所述融合用细胞系SP2/0。
7.根据权利要求6所述的脂联素单克隆抗体的制备方法，其特征在于，所述步骤3)中，所述脂联素重组蛋白免疫宿主动物包括以下步骤：以每只老鼠50ug脂联素重组蛋白加等量的弗氏完全佐剂，制成油包水乳剂，免疫6-8周龄Balb/C雌性小鼠；每两周免疫一次所述小鼠，每次免疫取50ug脂联素重组蛋白与弗氏不完全佐剂混匀；免疫四次后测效价，待效价达到1:10000以上时，按50ug/mouse腹腔冲击免疫。
8.根据权利要求2所述的脂联素单克隆抗体的制备方法，其特征在于，所述步骤4)中，所述获取免疫宿主动物的脾脏细胞采用以下步骤，腹腔冲击免疫三天后，在无菌条件下取出小鼠脾脏，并制成单个脾细胞悬液。
9.根据权利要求2所述的脂联素单克隆抗体的制备方法，其特征在于，所述步骤4)中，所述制备融合细胞工序，包括细胞融合和细胞建株，最后得到杂交瘤细胞。
10.根据权利要求9所述的脂联素单克隆抗体的制备方法，其特征在于，所述步骤4)中，所述制备融合细胞工序包括以下步骤：
步骤A，细胞融合：制备脾细胞悬液并用PBS洗涤干净后混入SP2/0细胞，脾细胞和SP2/0以10:1的比例混合并离心，离心后滴干混合细胞，轻敲弹松细胞团块，在37℃水浴中加入1ml PEG1450，加完后在37℃水浴中反应2min，沿管壁缓慢加入20ml RPMI-1640终止液，融合细胞终止后于800rpm离心5min，并吸干残留液体，HAT培养基重悬融合细胞，转入96孔细胞培养板培养，待融合细胞在HAT中培养7天左右后换成HT培养基；
步骤B，细胞建株：待孔中融合版细胞长至中等大小约104个细胞以上时开始检测，分别取50ul细胞培养液加入预先包被脂联素蛋白的微孔中，检测细胞上清抗体效价，从检测为阳性对应的微孔取阳性细胞进行亚克隆，利用HT培养基通过有限稀释法将筛选得到细胞稀释至1个/孔，将细胞铺于96孔细胞培养板，待单克隆细胞长至中等大小，密度约104个细胞以上即可检测效价，然后再次选取其中阳性细胞，重复一次亚克隆筛选，待检测到所有微孔中细胞上清都是阳性后，再进行一次同样的单克隆，直至有限稀释法后再次得到全阳性结果，能确定此时筛选到阳性细胞株，所得的阳性细胞株即为杂交瘤细胞。
11.根据权利要求2所述的脂联素单克隆抗体的制备方法，其特征在于，所述步骤5)中，融合细胞制备抗体，包括无血清制备和纯化步骤，纯化后得到不同的单克隆抗体。
12.根据权利要求11所述的脂联素单克隆抗体的制备方法，其特征在于，所述步骤5)中，无血清制备包括以下步骤：在有血清条件下复苏杂交瘤细胞，在连续几次细胞传代过程中，梯度减少血清的比例，逐渐增加代用的添加物，使杂交瘤细胞适应并继续生长；用无血清培养基复苏杂交瘤细胞，在细胞状态良好时，加大培养基的用量及培养细胞的容器，待细胞大量死亡时，收夜，并离心取上清，过滤收集，取待纯化的样品上样Protein A-琼脂糖亲和层析柱，流速为0.5ml/min，使抗体与Protein A结合，最后用洗脱液进行洗脱，得到抗体SDS-PAGE并鉴定其纯度。
13.脂联素单克隆抗体对，其特征在于：所述脂联素单克隆抗体对为CSB-DA120AmN③-CSB-DA120AmN④。
14.如权利要求13所述脂联素单克隆抗体对的制备方法，其特征在于，包括如下步骤：将所述的单克隆抗体CSB-DA120AmN③、CSB-DA120AmN④，分别用作捕获抗体和标记抗体，组合成抗体对用于双抗夹心法的免疫检测，将抗体对对各种浓度的脂联素重组蛋白进行检测，然后将单克隆抗体对应的细胞株提取RNA，经RT-PCR反转录得到cDNA，利用小鼠IgG抗体轻链和重链可变区通用引物进行DNA测序，确定抗体的可变区序列。
15.一种权利要求1和/或权利要求13所述的脂联素单克隆抗体和/或脂联素单克隆抗体对，在胶乳增强比浊、双抗夹心法的免疫检测产品中，或在制备检测脂联素的试剂、药剂或试剂盒中的应用。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011504233.7A CN112574306B (zh) | 2020-12-17 | 2020-12-17 | 脂联素单克隆抗体、抗体对及其制备方法和用途 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011504233.7A CN112574306B (zh) | 2020-12-17 | 2020-12-17 | 脂联素单克隆抗体、抗体对及其制备方法和用途 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112574306A true CN112574306A (zh) | 2021-03-30 |
| CN112574306B CN112574306B (zh) | 2023-05-05 |
Family
ID=75136089
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011504233.7A Active CN112574306B (zh) | 2020-12-17 | 2020-12-17 | 脂联素单克隆抗体、抗体对及其制备方法和用途 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112574306B (zh) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113671196A (zh) * | 2021-07-29 | 2021-11-19 | 中国人民解放军空军军医大学 | Lair-1分子与脂联素的相互作用对于t细胞活化作用影响的研究方法 |
| CN114149507A (zh) * | 2021-12-31 | 2022-03-08 | 武汉华美生物工程有限公司 | 抗fdp单克隆抗体、抗体对及其制备方法和用途 |
| CN116396384A (zh) * | 2023-01-17 | 2023-07-07 | 武汉爱博泰克生物科技有限公司 | 针对小鼠脂联素的兔单克隆抗体及其应用和双抗夹心法酶联免疫检测试剂盒 |
| CN117327177A (zh) * | 2022-06-24 | 2024-01-02 | 东莞市朋志生物科技有限公司 | 抗脂联素抗体、检测脂联素的试剂和试剂盒 |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003055916A2 (en) * | 2001-12-21 | 2003-07-10 | Maxygen Aps | Adiponectin fragments and conjugates |
| WO2004022596A1 (en) * | 2002-09-05 | 2004-03-18 | Komed Co., Ltd. | Monoclonal antibody against adiponectin, preparation method and use thereof |
| US20050074762A1 (en) * | 2001-02-01 | 2005-04-07 | Yusuke Nakamura | Adiponectin-associated protein |
| US20100111977A1 (en) * | 2008-10-15 | 2010-05-06 | 4S3 Bioscience Inc. | Methods and compositions for treatment of myotonic dystrophy |
| US20120070875A1 (en) * | 2009-05-28 | 2012-03-22 | Weisbart Richard H | Amino Acid Sequences which Enhance Peptide Conjugate Solubility |
| CN102517256A (zh) * | 2011-10-12 | 2012-06-27 | 陶凌 | 人脂联素elisa试剂盒 |
| CN104694478A (zh) * | 2014-11-06 | 2015-06-10 | 中国医学科学院北京协和医院 | 抗人脂联素的单克隆抗体及其应用 |
| CN106604740A (zh) * | 2014-02-14 | 2017-04-26 | 宏观基因有限公司 | 用于治疗血管化癌症的改进的方法 |
| CN110372783A (zh) * | 2019-04-15 | 2019-10-25 | 杭州博谱医药科技有限公司 | 一种脂联素抗原、抗体及其胶乳试剂的制备方法以及脂联素检测试剂盒 |
-
2020
- 2020-12-17 CN CN202011504233.7A patent/CN112574306B/zh active Active
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050074762A1 (en) * | 2001-02-01 | 2005-04-07 | Yusuke Nakamura | Adiponectin-associated protein |
| WO2003055916A2 (en) * | 2001-12-21 | 2003-07-10 | Maxygen Aps | Adiponectin fragments and conjugates |
| WO2004022596A1 (en) * | 2002-09-05 | 2004-03-18 | Komed Co., Ltd. | Monoclonal antibody against adiponectin, preparation method and use thereof |
| US20100111977A1 (en) * | 2008-10-15 | 2010-05-06 | 4S3 Bioscience Inc. | Methods and compositions for treatment of myotonic dystrophy |
| US20120070875A1 (en) * | 2009-05-28 | 2012-03-22 | Weisbart Richard H | Amino Acid Sequences which Enhance Peptide Conjugate Solubility |
| CN102517256A (zh) * | 2011-10-12 | 2012-06-27 | 陶凌 | 人脂联素elisa试剂盒 |
| CN106604740A (zh) * | 2014-02-14 | 2017-04-26 | 宏观基因有限公司 | 用于治疗血管化癌症的改进的方法 |
| CN104694478A (zh) * | 2014-11-06 | 2015-06-10 | 中国医学科学院北京协和医院 | 抗人脂联素的单克隆抗体及其应用 |
| CN110372783A (zh) * | 2019-04-15 | 2019-10-25 | 杭州博谱医药科技有限公司 | 一种脂联素抗原、抗体及其胶乳试剂的制备方法以及脂联素检测试剂盒 |
Non-Patent Citations (1)
| Title |
|---|
| 赵满仓等: "脂联素单链抗体基因的克隆及表达", 《河北医药》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113671196A (zh) * | 2021-07-29 | 2021-11-19 | 中国人民解放军空军军医大学 | Lair-1分子与脂联素的相互作用对于t细胞活化作用影响的研究方法 |
| CN113671196B (zh) * | 2021-07-29 | 2023-09-12 | 中国人民解放军空军军医大学 | Lair-1分子与脂联素的相互作用对于t细胞活化作用影响的研究方法 |
| CN114149507A (zh) * | 2021-12-31 | 2022-03-08 | 武汉华美生物工程有限公司 | 抗fdp单克隆抗体、抗体对及其制备方法和用途 |
| CN117327177A (zh) * | 2022-06-24 | 2024-01-02 | 东莞市朋志生物科技有限公司 | 抗脂联素抗体、检测脂联素的试剂和试剂盒 |
| CN116396384A (zh) * | 2023-01-17 | 2023-07-07 | 武汉爱博泰克生物科技有限公司 | 针对小鼠脂联素的兔单克隆抗体及其应用和双抗夹心法酶联免疫检测试剂盒 |
| CN116396384B (zh) * | 2023-01-17 | 2023-11-17 | 武汉爱博泰克生物科技有限公司 | 针对小鼠脂联素的兔单克隆抗体及其应用和双抗夹心法酶联免疫检测试剂盒 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112574306B (zh) | 2023-05-05 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN112574306A (zh) | 脂联素单克隆抗体、抗体对及其制备方法和用途 | |
| CN111499746B (zh) | 一种针对人白介素-2的高亲和力兔单克隆抗体及其应用 | |
| CN113150136B (zh) | 新型冠状病毒n蛋白单克隆抗体的制备 | |
| CN112778416B (zh) | 一种纳米抗体、涉及该纳米抗体的多肽及其应用 | |
| CN110845582B (zh) | 一种猫细小病毒重组蛋白及其单克隆抗体的制备 | |
| CN112500479B (zh) | 一种犬ⅱ型腺病毒重组蛋白单克隆抗体的制备 | |
| CN105542010B (zh) | 脂蛋白相关磷脂酶a2单克隆抗体、抗体对及制备方法、用途 | |
| CN112608385B (zh) | 一种犬脑钠肽(bnp)单克隆抗体的制备 | |
| CN113150138B (zh) | 一种kpc-2单克隆抗体及其制备方法和应用 | |
| CN109142738A (zh) | Ecm1作为血清学检测肝纤维化的标志物及其应用 | |
| CN116925218A (zh) | 小热休克蛋白hspb1的抗体、抗体组合物、杂交瘤细胞株及其应用 | |
| CN115819580A (zh) | 针对人il-12的高亲和力兔单克隆抗体及其应用 | |
| CN104558145B (zh) | 一种胎球蛋白a重组蛋白及多克隆抗体的制备方法 | |
| CN110903359B (zh) | 一种空肠弯曲菌重组蛋白及其单克隆抗体的制备 | |
| CN115052899B (zh) | Pcsk9的单域抗体及其应用 | |
| CN112661849A (zh) | 一种艰难梭菌重组蛋白单克隆抗体的制备方法和应用 | |
| US20080220538A1 (en) | Novel Cancer Associated Antibodies And Their Use In Cancer Diagnosis | |
| CN112111496A (zh) | 稀有鮈鲫载脂蛋白ApoE基因、重组蛋白、多克隆抗体及制备方法和应用 | |
| CN110951703A (zh) | 一种间日疟原虫乳酸脱氢酶重组蛋白及其单克隆抗体的制备 | |
| CN116925219B (zh) | 小热休克蛋白hspb1的抗体、杂交瘤细胞株及其应用 | |
| CN112763723A (zh) | 检测血清thbs2表达的肝病纤维化检测试剂、检测试剂盒和检测方法 | |
| CN115948346A (zh) | 一株能稳定分泌抗芋螺毒素μ-KIIIA-CTX单克隆抗体的杂交瘤细胞株 | |
| CN115057935A (zh) | 抗人弹性蛋白酶抑制蛋白抗体、试剂盒及其制备方法和应用 | |
| CN116813742A (zh) | 人gdf-15抗原表位肽、抗原、抗体、试剂盒及应用 | |
| CN117736293A (zh) | 一种猪crisp3蛋白及其单克隆抗体的制备方法及应用 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: Adiponectin monoclonal antibodies, antibody pairs, preparation methods and applications Granted publication date: 20230505 Pledgee: Guanggu Branch of Wuhan Rural Commercial Bank Co.,Ltd. Pledgor: CUSABIO BIOTECH Co.,Ltd. Registration number: Y2024980009781 |