CN105524145B - 一种高效识别蛋白精氨酸鼠李糖基化的多克隆抗体及其制备方法 - Google Patents
一种高效识别蛋白精氨酸鼠李糖基化的多克隆抗体及其制备方法 Download PDFInfo
- Publication number
- CN105524145B CN105524145B CN201510891290.8A CN201510891290A CN105524145B CN 105524145 B CN105524145 B CN 105524145B CN 201510891290 A CN201510891290 A CN 201510891290A CN 105524145 B CN105524145 B CN 105524145B
- Authority
- CN
- China
- Prior art keywords
- compound
- arginine
- rhamnopyranosylization
- modification
- glycopeptide
- 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.)
- Active
Links
- 239000004475 Arginine Substances 0.000 title claims abstract description 46
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 title claims abstract description 43
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 17
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 17
- 238000002360 preparation method Methods 0.000 title claims abstract description 10
- 230000004048 modification Effects 0.000 claims abstract description 29
- 238000012986 modification Methods 0.000 claims abstract description 29
- 238000000034 method Methods 0.000 claims abstract description 13
- 239000000427 antigen Substances 0.000 claims abstract description 12
- 108091007433 antigens Proteins 0.000 claims abstract description 12
- 102000036639 antigens Human genes 0.000 claims abstract description 12
- 238000001514 detection method Methods 0.000 claims abstract description 9
- 239000007790 solid phase Substances 0.000 claims abstract description 9
- 230000003115 biocidal effect Effects 0.000 claims abstract description 7
- 238000006555 catalytic reaction Methods 0.000 claims abstract description 7
- 238000012216 screening Methods 0.000 claims abstract description 6
- 230000008569 process Effects 0.000 claims abstract description 5
- 241001465754 Metazoa Species 0.000 claims abstract description 3
- 150000001875 compounds Chemical class 0.000 claims description 29
- DQJCDTNMLBYVAY-ZXXIYAEKSA-N (2S,5R,10R,13R)-16-{[(2R,3S,4R,5R)-3-{[(2S,3R,4R,5S,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy}-5-(ethylamino)-6-hydroxy-2-(hydroxymethyl)oxan-4-yl]oxy}-5-(4-aminobutyl)-10-carbamoyl-2,13-dimethyl-4,7,12,15-tetraoxo-3,6,11,14-tetraazaheptadecan-1-oic acid Chemical compound NCCCC[C@H](C(=O)N[C@@H](C)C(O)=O)NC(=O)CC[C@H](C(N)=O)NC(=O)[C@@H](C)NC(=O)C(C)O[C@@H]1[C@@H](NCC)C(O)O[C@H](CO)[C@H]1O[C@H]1[C@H](NC(C)=O)[C@@H](O)[C@H](O)[C@@H](CO)O1 DQJCDTNMLBYVAY-ZXXIYAEKSA-N 0.000 claims description 25
- 108010015899 Glycopeptides Proteins 0.000 claims description 24
- 102000002068 Glycopeptides Human genes 0.000 claims description 24
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 24
- 238000011068 loading method Methods 0.000 claims description 15
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims description 12
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 12
- DYHSDKLCOJIUFX-UHFFFAOYSA-N tert-butoxycarbonyl anhydride Chemical compound CC(C)(C)OC(=O)OC(=O)OC(C)(C)C DYHSDKLCOJIUFX-UHFFFAOYSA-N 0.000 claims description 12
- 102000014914 Carrier Proteins Human genes 0.000 claims description 11
- 108010078791 Carrier Proteins Proteins 0.000 claims description 11
- 238000000746 purification Methods 0.000 claims description 11
- 229920002684 Sepharose Polymers 0.000 claims description 9
- 230000009471 action Effects 0.000 claims description 9
- 238000010828 elution Methods 0.000 claims description 9
- 239000007788 liquid Substances 0.000 claims description 8
- SQGYOTSLMSWVJD-UHFFFAOYSA-N silver(1+) nitrate Chemical compound [Ag+].[O-]N(=O)=O SQGYOTSLMSWVJD-UHFFFAOYSA-N 0.000 claims description 8
- 239000000945 filler Substances 0.000 claims description 7
- NWZSZGALRFJKBT-KNIFDHDWSA-N (2s)-2,6-diaminohexanoic acid;(2s)-2-hydroxybutanedioic acid Chemical compound OC(=O)[C@@H](O)CC(O)=O.NCCCC[C@H](N)C(O)=O NWZSZGALRFJKBT-KNIFDHDWSA-N 0.000 claims description 6
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 6
- IVLXQGJVBGMLRR-UHFFFAOYSA-N 2-aminoacetic acid;hydron;chloride Chemical compound Cl.NCC(O)=O IVLXQGJVBGMLRR-UHFFFAOYSA-N 0.000 claims description 6
- -1 9-fluorenylmethyloxycarbonyl Chemical group 0.000 claims description 6
- VGCXGMAHQTYDJK-UHFFFAOYSA-N Chloroacetyl chloride Chemical compound ClCC(Cl)=O VGCXGMAHQTYDJK-UHFFFAOYSA-N 0.000 claims description 6
- 229910021529 ammonia Inorganic materials 0.000 claims description 6
- 229940126214 compound 3 Drugs 0.000 claims description 6
- 229940125898 compound 5 Drugs 0.000 claims description 6
- 238000004132 cross linking Methods 0.000 claims description 6
- 239000012149 elution buffer Substances 0.000 claims description 6
- 239000006167 equilibration buffer Substances 0.000 claims description 6
- IKDUDTNKRLTJSI-UHFFFAOYSA-N hydrazine monohydrate Substances O.NN IKDUDTNKRLTJSI-UHFFFAOYSA-N 0.000 claims description 6
- 238000010992 reflux Methods 0.000 claims description 6
- 239000011347 resin Substances 0.000 claims description 6
- 229920005989 resin Polymers 0.000 claims description 6
- 239000011780 sodium chloride Substances 0.000 claims description 6
- DPKBAXPHAYBPRL-UHFFFAOYSA-M tetrabutylazanium;iodide Chemical compound [I-].CCCC[N+](CCCC)(CCCC)CCCC DPKBAXPHAYBPRL-UHFFFAOYSA-M 0.000 claims description 6
- 238000006243 chemical reaction Methods 0.000 claims description 5
- 229910052801 chlorine Inorganic materials 0.000 claims description 5
- 239000000460 chlorine Substances 0.000 claims description 5
- 230000000694 effects Effects 0.000 claims description 5
- HVTICUPFWKNHNG-UHFFFAOYSA-N iodoethane Chemical compound CCI HVTICUPFWKNHNG-UHFFFAOYSA-N 0.000 claims description 5
- ZNNZYHKDIALBAK-UHFFFAOYSA-M potassium thiocyanate Chemical compound [K+].[S-]C#N ZNNZYHKDIALBAK-UHFFFAOYSA-M 0.000 claims description 5
- RXLIOYNXBHZZBI-NRFANRHFSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-5-(prop-2-enoxycarbonylamino)pentanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CCCNC(=O)OCC=C)C(=O)O)C3=CC=CC=C3C2=C1 RXLIOYNXBHZZBI-NRFANRHFSA-N 0.000 claims description 4
- 150000001413 amino acids Chemical class 0.000 claims description 4
- 238000004176 ammonification Methods 0.000 claims description 4
- 239000003431 cross linking reagent Substances 0.000 claims description 4
- 238000010647 peptide synthesis reaction Methods 0.000 claims description 4
- 229910001961 silver nitrate Inorganic materials 0.000 claims description 4
- ULARYIUTHAWJMU-UHFFFAOYSA-M sodium;1-[4-(2,5-dioxopyrrol-1-yl)butanoyloxy]-2,5-dioxopyrrolidine-3-sulfonate Chemical compound [Na+].O=C1C(S(=O)(=O)[O-])CC(=O)N1OC(=O)CCCN1C(=O)C=CC1=O ULARYIUTHAWJMU-UHFFFAOYSA-M 0.000 claims description 4
- 229940125904 compound 1 Drugs 0.000 claims description 3
- 229940125782 compound 2 Drugs 0.000 claims description 3
- 150000003384 small molecules Chemical class 0.000 claims description 2
- 230000007935 neutral effect Effects 0.000 claims 1
- 238000005303 weighing Methods 0.000 claims 1
- 102000004196 processed proteins & peptides Human genes 0.000 abstract description 9
- 108090000765 processed proteins & peptides Proteins 0.000 abstract description 9
- 229920001184 polypeptide Polymers 0.000 abstract description 7
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 abstract description 4
- 229910052709 silver Inorganic materials 0.000 abstract description 4
- 239000004332 silver Substances 0.000 abstract description 4
- 238000001261 affinity purification Methods 0.000 abstract description 3
- 210000004369 blood Anatomy 0.000 abstract description 3
- 239000008280 blood Substances 0.000 abstract description 3
- 241000283977 Oryctolagus Species 0.000 abstract description 2
- 229940125644 antibody drug Drugs 0.000 abstract description 2
- 229940126586 small molecule drug Drugs 0.000 abstract description 2
- 230000021615 conjugation Effects 0.000 abstract 1
- 235000009697 arginine Nutrition 0.000 description 29
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 12
- 235000018102 proteins Nutrition 0.000 description 11
- 239000000243 solution Substances 0.000 description 10
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 9
- 108010056732 factor EF-P Proteins 0.000 description 9
- 230000013595 glycosylation Effects 0.000 description 9
- 238000006206 glycosylation reaction Methods 0.000 description 9
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 6
- 238000003119 immunoblot Methods 0.000 description 5
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 4
- 238000003556 assay Methods 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 210000004027 cell Anatomy 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 210000002966 serum Anatomy 0.000 description 4
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 description 3
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 3
- SHZGCJCMOBCMKK-JFNONXLTSA-N L-rhamnopyranose Chemical compound C[C@@H]1OC(O)[C@H](O)[C@H](O)[C@H]1O SHZGCJCMOBCMKK-JFNONXLTSA-N 0.000 description 3
- PNNNRSAQSRJVSB-UHFFFAOYSA-N L-rhamnose Natural products CC(O)C(O)C(O)C(O)C=O PNNNRSAQSRJVSB-UHFFFAOYSA-N 0.000 description 3
- 239000006180 TBST buffer Substances 0.000 description 3
- 239000002671 adjuvant Substances 0.000 description 3
- 238000004945 emulsification Methods 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 210000003705 ribosome Anatomy 0.000 description 3
- 238000007920 subcutaneous administration Methods 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 2
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 2
- KDLHZDBZIXYQEI-UHFFFAOYSA-N Palladium Chemical compound [Pd] KDLHZDBZIXYQEI-UHFFFAOYSA-N 0.000 description 2
- 241000589517 Pseudomonas aeruginosa Species 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
- 108090000992 Transferases Proteins 0.000 description 2
- 102000004357 Transferases Human genes 0.000 description 2
- 235000001014 amino acid Nutrition 0.000 description 2
- 229940024606 amino acid Drugs 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000004140 cleaning Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 244000052637 human pathogen Species 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 230000001717 pathogenic effect Effects 0.000 description 2
- 238000000197 pyrolysis Methods 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 239000013589 supplement Substances 0.000 description 2
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 2
- UMGDCJDMYOKAJW-UHFFFAOYSA-N thiourea Chemical compound NC(N)=S UMGDCJDMYOKAJW-UHFFFAOYSA-N 0.000 description 2
- ZGYICYBLPGRURT-UHFFFAOYSA-N tri(propan-2-yl)silicon Chemical compound CC(C)[Si](C(C)C)C(C)C ZGYICYBLPGRURT-UHFFFAOYSA-N 0.000 description 2
- RIOQSEWOXXDEQQ-UHFFFAOYSA-N triphenylphosphine Chemical compound C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1 RIOQSEWOXXDEQQ-UHFFFAOYSA-N 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 description 1
- GEYOCULIXLDCMW-UHFFFAOYSA-N 1,2-phenylenediamine Chemical compound NC1=CC=CC=C1N GEYOCULIXLDCMW-UHFFFAOYSA-N 0.000 description 1
- PXFBZOLANLWPMH-UHFFFAOYSA-N 16-Epiaffinine Natural products C1C(C2=CC=CC=C2N2)=C2C(=O)CC2C(=CC)CN(C)C1C2CO PXFBZOLANLWPMH-UHFFFAOYSA-N 0.000 description 1
- MMHMYFWOECSGDR-UHFFFAOYSA-N 2,5-dimethoxybenzenesulfonamide Chemical compound COC1=CC=C(OC)C(S(N)(=O)=O)=C1 MMHMYFWOECSGDR-UHFFFAOYSA-N 0.000 description 1
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 206010010356 Congenital anomaly Diseases 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 102000051366 Glycosyltransferases Human genes 0.000 description 1
- 108700023372 Glycosyltransferases Proteins 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 239000007836 KH2PO4 Substances 0.000 description 1
- 235000019766 L-Lysine Nutrition 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-N L-arginine Chemical compound OC(=O)[C@@H](N)CCCN=C(N)N ODKSFYDXXFIFQN-BYPYZUCNSA-N 0.000 description 1
- 229930064664 L-arginine Natural products 0.000 description 1
- 235000014852 L-arginine Nutrition 0.000 description 1
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 1
- DUKURNFHYQXCJG-UHFFFAOYSA-N Lewis A pentasaccharide Natural products OC1C(O)C(O)C(C)OC1OC1C(OC2C(C(O)C(O)C(CO)O2)O)C(NC(C)=O)C(OC2C(C(OC3C(OC(O)C(O)C3O)CO)OC(CO)C2O)O)OC1CO DUKURNFHYQXCJG-UHFFFAOYSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- NMOJAXCSURVGEY-UHFFFAOYSA-N N#CC#N.[S] Chemical compound N#CC#N.[S] NMOJAXCSURVGEY-UHFFFAOYSA-N 0.000 description 1
- OVRNDRQMDRJTHS-FMDGEEDCSA-N N-acetyl-beta-D-glucosamine Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-FMDGEEDCSA-N 0.000 description 1
- 230000004988 N-glycosylation Effects 0.000 description 1
- 241000588653 Neisseria Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 201000005702 Pertussis Diseases 0.000 description 1
- 229920000037 Polyproline Polymers 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- 241001223867 Shewanella oneidensis Species 0.000 description 1
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 235000009754 Vitis X bourquina Nutrition 0.000 description 1
- 235000012333 Vitis X labruscana Nutrition 0.000 description 1
- 240000006365 Vitis vinifera Species 0.000 description 1
- 235000014787 Vitis vinifera Nutrition 0.000 description 1
- 230000001594 aberrant effect Effects 0.000 description 1
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 description 1
- 230000004721 adaptive immunity Effects 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- PNNNRSAQSRJVSB-BXKVDMCESA-N aldehydo-L-rhamnose Chemical compound C[C@H](O)[C@H](O)[C@@H](O)[C@@H](O)C=O PNNNRSAQSRJVSB-BXKVDMCESA-N 0.000 description 1
- 150000001335 aliphatic alkanes Chemical class 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 238000011091 antibody purification Methods 0.000 description 1
- 230000006253 arginine-glycosylation Effects 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- SHZGCJCMOBCMKK-YJRYQGEOSA-N beta-L-rhamnopyranose Chemical compound C[C@@H]1O[C@H](O)[C@H](O)[C@H](O)[C@H]1O SHZGCJCMOBCMKK-YJRYQGEOSA-N 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 238000005034 decoration Methods 0.000 description 1
- 230000007123 defense Effects 0.000 description 1
- 230000007850 degeneration Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 229930182470 glycoside Natural products 0.000 description 1
- 125000003147 glycosyl group Chemical group 0.000 description 1
- 102000035122 glycosylated proteins Human genes 0.000 description 1
- 108091005608 glycosylated proteins Proteins 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 239000000543 intermediate Substances 0.000 description 1
- 229910052740 iodine Inorganic materials 0.000 description 1
- 239000011630 iodine Substances 0.000 description 1
- 150000002540 isothiocyanates Chemical class 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 229950006780 n-acetylglucosamine Drugs 0.000 description 1
- XRRQZKOZJFDXON-UHFFFAOYSA-N nitric acid;silver Chemical compound [Ag].O[N+]([O-])=O XRRQZKOZJFDXON-UHFFFAOYSA-N 0.000 description 1
- 229910052763 palladium Inorganic materials 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 108010026466 polyproline Proteins 0.000 description 1
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 125000001500 prolyl group Chemical group [H]N1C([H])(C(=O)[*])C([H])([H])C([H])([H])C1([H])[H] 0.000 description 1
- UIDUKLCLJMXFEO-UHFFFAOYSA-N propylsilane Chemical compound CCC[SiH3] UIDUKLCLJMXFEO-UHFFFAOYSA-N 0.000 description 1
- 230000009145 protein modification Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000004007 reversed phase HPLC Methods 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 150000007970 thio esters Chemical class 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000005029 transcription elongation Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K9/00—Peptides having up to 20 amino acids, containing saccharide radicals and having a fully defined sequence; Derivatives thereof
- C07K9/001—Peptides having up to 20 amino acids, containing saccharide radicals and having a fully defined sequence; Derivatives thereof the peptide sequence having less than 12 amino acids and not being part of a ring structure
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/765—Serum albumin, e.g. HSA
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
- C07K16/065—Purification, fragmentation
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- Toxicology (AREA)
- Zoology (AREA)
- Gastroenterology & Hepatology (AREA)
- Peptides Or Proteins (AREA)
Abstract
本发明涉及一种高效识别蛋白精氨酸鼠李糖基化多克隆抗体的制备方法,该抗体主要用于包含精氨酸鼠李糖基化修饰的天然蛋白的检测以及新型抗生素的筛选与发现。过程:第一步,在银催化下的固相条件下直接进行糖基化得到包含精氨酸鼠李糖基化的多肽,其序列为:Ac‑CGR(Rha)GL‑OH。第二步,将得到的半抗原偶联到载体蛋白BSA上,得到抗原,并将其免疫动物,收集免疫新西兰兔血液制备抗血清,通过亲和纯化得到IgG。本发明的抗体生产成本低,特异性好,效价高,能特异性地识别精氨酸鼠李糖基化修饰的蛋白。该抗体可用于发现更多的包含精氨酸鼠李糖基化修饰的天然蛋白,以及包含抗体药物以及小分子药物两方面的新型抗生素的筛选。
Description
技术领域
本发明涉及医药化学技术领域,具体地说,是一种高效识别蛋白精氨酸鼠李糖基化的多克隆抗体及其制备方法。
背景技术
糖基化修饰是一种生物系统中最重要的蛋白翻译后修饰,异常糖基化通常是与许多生物过程(包括病毒和细菌感染、癌症转移、炎症反应、先天和适应性免疫)及其它信号通路有着密切联系的。在很长一段时间内,蛋白质糖基化修饰被认为局限于真核生物,而今天人们广泛接受了包括重要的人类病原体在内细胞当中也含有大量的O-以及N-糖基化修饰。其中关于N-糖基化修饰的报道似乎仅仅发生于天冬酰胺上,直到2013年,两个独立的研究小组才发现了首个精氨酸鼠李糖基化的修饰,他们同时发现致病大肠杆菌(EPEC)中III型分泌系统因子NleB,能够作为精氨酸乙酰氨基葡萄糖(ArgGlcNAc)转移酶在人类死亡受体域进行糖基化修饰,从而干扰宿主防御。最近,我们发现另一种非常重要的精氨酸糖基化修饰,该糖基化修饰能够激活转录延长因子EF-P,从而缓解核糖体在多聚脯氨酸序列附件的停滞(图1)。某些细菌包括重要的人类病原体,如绿脓杆菌、百日咳菌、淋病奈瑟氏菌属,为了实现这种核糖体的解救,能够特异性地实现32位精氨酸的鼠李糖基化修饰。当EF-P绑定到核糖体之后,其鼠李糖基化修饰的精氨酸能够到达转肽酶中心为包含脯氨酸转移酶的tRNA提供有利位置,从而稳定了脯氨酰-tRNA的CCA末端。鼠李糖基化修饰破坏绿脓杆菌的致病性,从而一定程度上增加了抗生素的作用。因此抑制EF-P的鼠李糖基化修饰可能成为一种选择性地抑制毒性的新策略。
2014年,我们成功地合成了含有精氨酸氨基葡萄糖基化的多肽,并成功制备了高亲和力,高特异性的抗精氨酸氨基葡萄糖基化的抗体(Pan M etal,Synthesis of andSpecific Antibody Generation for Glycopeptides with Arginine N-GlcNAcylation,Angew.Chem.Int.Ed.2014,53,14517-14521)。虽然该抗体能够精确检测包含精氨酸氨基葡萄糖基化的蛋白质,但其不能检测包含EF-P在内的精氨酸鼠李糖基化修饰的蛋白。因此我们第一步在银催化下的固相条件下直接进行糖基化得到包含精氨酸鼠李糖基化的多肽。其次我们将通过该糖肽的氨基端巯基与牛血清白蛋白(BSA)偶联,最后用它作为抗原免疫兔子,将得到的抗血清进行亲和纯化,从而得到首个特异性地抗精氨酸鼠李糖基化的抗体(Anti-ArgRha)(图2)。
发明内容
本发明的目的是针对现有技术中的不足,提供一种含有精氨酸鼠李糖基化修饰的糖肽。
本发明的再一的目的是,提供如上所述糖肽的制备方法。
本发明的另一的目的是,提供如上所述糖肽与载体蛋白交联的化合物。
本发明的第四个的目的是,提供特异性识别如上所述糖肽的抗血清或抗体。
本发明的第五个目的是,提供如上所述抗体的纯化方法。
本发明的第六个目的是,提供如上所述抗体的用途。
为实现上述目的,本发明采取的技术方案是:
一种含有精氨酸鼠李糖基化修饰的糖肽,所述糖肽结构如下所示:
为实现上述第二个目的,本发明采取的技术方案是:
如上所述含有精氨酸鼠李糖基化修饰的糖肽的制备方法,包括如下步骤:
a)化合物2在乙酰氯中反应得到化合物3;
b)化合物3在硫氰化钾与四丁基碘化铵的作用下回流得到化合物4;
c)化合物4在氨气作用下氨化得到化合物5;
d)化合物5在碘乙烷、二碳酸二叔丁酯、三乙胺的作用下分两步反应得到化合物6;
e)以2-氯三苯甲基树脂作为载体,Fmoc-Orn(Alloc)-OH作为糖基化的启动氨基酸,采用基于9-芴甲氧羰基的固相肽合成方法制备化合物7;
f)化合物7与化合物6在硝酸银催化下反应得到化合物8;
g)化合物8在5%的水合肼作用脱去乙酰基,得到目标化合物1;
试剂与条件:a)乙酰氯,室温,两天,85%;b)硫氰化钾,四丁基碘化铵,乙腈,回流,3小时,70%;c)氨气,四氢呋喃,1小时,99%;d)碘乙烷,甲醇,回流3小时;二碳酸二叔丁酯,三乙胺,二氯甲烷,75%。
试剂与条件:a)三乙胺,硝酸银,N,N-二甲基甲酰胺,6(3eq.),室温;
b)5%水合肼/N,N-二甲基甲酰胺;c)5%三异丙基硅烷/三氟乙酸。
为实现上述第三个目的,本发明采取的技术方案是:
如上所述含有精氨酸鼠李糖基化修饰的糖肽与载体蛋白交联的化合物。
优选地,所述的载体蛋白为BSA,所述的交联为采用Sulfo-GMBS作为交联剂将载体蛋白BSA与糖肽N-端的巯基交联。
为实现上述第四个目的,本发明采取的技术方案是:
特异性识别如上所述含有精氨酸鼠李糖基化修饰的糖肽的抗血清或抗体。
为实现上述第五个目的,本发明采取的技术方案是:
如上所述抗体的纯化方法,包括如下步骤:
a)Protein A纯化:纯化系统:AKTA purifier 10(Amersham Biosciences);填料:Protein A Sepharose 4 Fast Flow;平衡缓冲液:20mM PBS,0.15M NaCl,pH7.2;洗脱缓冲液:0.1M glycine-HCl,pH2.8;上样流速:1ml/min,流程:平衡柱子→上样→再平衡→洗脱,洗脱后样品用1M Tris-HCl(pH=9)调pH至中性,PBS透析过夜;
b)特异性纯化:纯化系统:AKTA purifier 10(Amersham Biosciences);填料:BSASepharose 4 Fast Flow&CL5(SH)-BSA Sepharose 4 Fast Flow;平衡缓冲液:20mM PBS,0.15M NaCl,pH7.2;洗脱缓冲液:0.1M glycine-HCl(pH==2.8);上样流速:1ml/min;流程:平衡柱子→上样→收集流穿→再上样→收集流穿,洗脱后样品用1M Tris-HCl(pH=9)调pH至中性。
为实现上述第六个目的,本发明采取的技术方案是:
如上所述的抗体在检测EF-P的精氨酸鼠李糖修饰上的应用。
如上所述的抗体在检测其它精氨酸鼠李糖基化修饰的蛋白中的应用。
如上所述的抗体在制备抗体类抗生素中的应用。
如上的抗体在小分子抗生素筛选中的应用。
本发明优点在于:
本发明涉及一种高效识别蛋白精氨酸鼠李糖基化多克隆抗体的制备方法,这种抗体主要用于包含精氨酸鼠李糖基化修饰的天然蛋白的检测以及新型抗生素的筛选与发现。其主要有以下过程:第一步,我们利用在银催化下的固相条件下直接进行糖基化得到包含精氨酸鼠李糖基化的多肽,其序列为:Ac-CGR(Rha)GL-OH。第二步,我们将得到的半抗原偶联到载体蛋白BSA上,得到抗原,并将其免疫动物,收集免疫新西兰兔血液制备抗血清,并通过亲和纯化得到IgG。本发明制备的抗体具有生产成本低,特异性好,效价高,已证实能够特异性地识别精氨酸鼠李糖基化修饰的蛋白,而不识别没有精氨酸鼠李糖基化修饰的蛋白以及其他的N-糖基化修饰的蛋白。该抗体能够用于发现更多的包含精氨酸鼠李糖基化修饰的天然蛋白,以及包含抗体药物以及小分子药物两方面的新型抗生素的筛选。
附图说明
附图1是EF-P的精氨酸鼠李糖基化修饰模式图。
附图2是精氨酸鼠李糖基化的多肽的合成以及抗精氨酸鼠李糖基化的抗体的制备示意图。
附图3是关键中间体6的合成路线图。试剂与条件:a)乙酰氯,室温,两天,85%;b)硫氰化钾,四丁基碘化铵,乙腈,回流,3小时,70%;c)氨气,四氢呋喃,1小时,99%;d)碘乙烷,甲醇,回流3小时;二碳酸二叔丁酯,三乙胺,二氯甲烷,75%。
附图4是精氨酸鼠李糖基化的多肽1的合成路线图。试剂与条件:a)三乙胺,硝酸银,N,N-二甲基甲酰胺,中间体6(3eq.),室温;b)5%水合肼/N,N-二甲基甲酰胺;c)5%三异丙基硅烷/三氟乙酸。
附图5是间接ELISA法测定粗血清与纯化后抗体的效价。
附图6是Anti-ArgRha抗体特异性识别鼠李糖基化修饰以及检测限的确定。
附图7是Anti-ArgRha抗体特异性地确定。
附图8是细胞裂解液中精氨酸鼠李糖基化的检测。
具体实施方式
下面结合具体实施方式,进一步阐述本发明。应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。此外应理解,在阅读了本发明讲授的内容之后,本领域技术人员可以对本发明作各种改动或修改,这些等价形式同样落于本申请所附权利要求书所限定的范围。
实施例1关键中间体6的合成
首先以L-鼠李糖2为原料,在乙酰氯的条件下得到三乙酰保护的α-氯苷3,3在硫氰化钾与四丁基碘化铵的作用下在乙腈中回流得到鼠李糖的异硫氰酸酯4,随后4在氨气的作用下氨化得到鼠李糖硫脲5,最后5在碘乙烷,二碳酸二叔丁酯,三乙胺的作用下分两步反应得到关键中间体6。具体合成路线见图3。
实施例2精氨酸鼠李糖基化的多肽1的合成
采用固相糖基化策略,具体合成路线见图4。首先直链肽采用基于9-芴甲氧羰基(Fmoc)的固相肽合成方法(SPPS)。2-氯三苯甲基树脂作为载体。Fmoc-Orn(Alloc)-OH作为糖基化的启动氨基酸。得到直链肽后,我们在(三苯基膦)钯的作用下切去侧链氨基的保护基得到化合物7。然后在树脂上利用银催化下的氨基与硫酯的反应得到糖肽10。接下来,鼠李糖上的乙酰保护基在5%的水合肼作用下脱去。最后树脂用5%三异丙基硅烷/三氟乙酸切割,进一步通过反相高效液相制备色谱得到纯的糖肽1。总收率为28%,展现良好的固相糖基化效率。
实施例3糖肽与载体蛋白交联
采用Sulfo-GMBS作为交联剂将载体蛋白BSA与糖肽N-端的巯基交联,具体方法如下:将20mg BSA溶于2ml 10mM K2HPO4-KH2PO4的缓冲液中,加入420ul MBS(3mg/ml),反应半小时;将20mg 1溶于1ml水中,缓慢滴入BSA溶液中,调节pH至7.4,室温反应2小时,PBS透析,待用。
实施例4糖肽免疫与抗血清制备
1、用抗原与Freund’s佐剂混合,乳化后,皮下和皮内多点注射进行免疫。抗原的剂量约为1mg/次。
2、间隔时间为3周。
3、再次免疫用不完全Freund’s佐剂,乳化后,皮下和皮内多点注射进行免疫。抗原的剂量约为1g/次。
4、间隔时间为3周。
5、三次免疫用不完全Freund’s佐剂,乳化后,皮下和皮内多点注射进行免疫。抗原的剂量约为1mg/次。
6、第三次免疫后1周,抽血,收集抗血清。
7、次日分离血清,-20℃保存。
实施例5抗体的纯化
采用亲和层析的方法纯化抗体,分别利用Protein A填料和BSA Sepharose 4以及CL5(SH)-BSA Sepharose 4三次亲和柱纯化。
1、Protein A纯化:纯化系统:AKTA purifier 10(Amersham Biosciences),填料:Protein A Sepharose 4 Fast Flow,平衡缓冲液:20mM PBS,0.15M NaCl,pH7.2,洗脱缓冲液:0.1M glycine-HCl,pH2.8,上样流速:1ml/min,流程:平衡柱子→上样→再平衡→洗脱,洗脱后样品用1M Tris-HCl(pH=9)调pH至中性,PBS透析过夜。
2、特异性纯化:纯化系统:AKTA purifier 10(Amersham Biosciences),填料:BSASepharose 4 Fast Flow&CL5(SH)-BSA Sepharose 4 Fast Flow,平衡缓冲液:20mM PBS,0.15M NaCl,pH7.2,洗脱缓冲液:0.1M glycine-HCl(pH==2.8),上样流速:1ml/min,流程:平衡柱子→上样→收集流穿→再上样→收集流穿,洗脱后样品用1M Tris-HCl(pH=9)调pH至中性。
实施例6间接ELISA法测定抗血清以及纯化后抗体的效价
1.包被抗原:将BSA或者BSA-1(5μg/mL)溶于包被液中(0.032M Na2CO3 and 0.068MNaHCO3,pH 9.6)。100μL样品加入96孔板中,37℃温育1小时后,4℃冰箱放置16~18小时。
2.洗涤:倒尽板孔中液体,加满洗涤液,静放三分钟,反复三次,最后将反应板倒置在吸水纸上,使孔中洗涤液流尽。
3.加封闭液200微升,37℃放置一小时。
4.洗涤同2。
5.加被检血清:用稀释液将被检血清稀释,每孔200微升。同时作稀释液对照。37℃放置2小时。
6.洗涤同2。
7.加辣根过氧化物酶羊抗兔IgG,每孔200微升,放置37℃1小时。
8.洗涤同2。
9.加底物:邻苯二胺溶液加200ml,室温暗处10-15分钟。
10.加终止液:每孔50微升。
11.观察结果:用酶联免疫检测仪记录490nm读数,结果见图5。
实施例7免疫印迹实验确证抗体的特异性
按照标准方法配置SDS-PAGE凝胶,调整样品蛋白浓度后,和等体积2*上样缓冲液混合后于95℃水中煮10分钟,蛋白变性后离心1分钟(3000rpm/min)。200V恒压电泳20分钟。停止电泳后,采用电转膜法恒流300mA 120分钟转膜至PVDF膜,用3%BSA室温封闭1小时,于摇床上用TBST洗膜三次。将实施例五中获得的抗体用TBST稀释至所需浓度后加入孵育袋中,4℃孵育过夜。TBST洗膜三次后辣根过氧化物(HRP)标记的抗兔二抗室温孵育2小时后,用Gel DocTM(BioRad)进行免疫检测与定量。
1.Anti-ArgRha抗体特异性识别鼠李糖基化修饰:使用0.5μg纯化后的糖基化修饰或未修饰的EF-P,进行SDS-PAGE及免疫印迹分析。
2.抗体检测限的确定:最小检测抗体浓度测定采用0.5μg EF-PRha和Anti-ArgRha抗体(2μg/ml,1μg/ml,0.4μg/ml,0.2μg/ml,0.1μg/ml,0.04μg/ml,0.02μg/ml)。
3.抗原检测极限的确定:EF-PRha(0.5μg,0.25μg,0.125μg,62.5ng,31.25ng,15.6ng,7.8ng,3.9ng)进行SDS-PAGE及免疫印迹分析。抗体两种浓度:2μg/ml和0.2μg/ml。
4.交叉免疫原性的排除:分别用EF-PRha(0.015μM,0.15μM,1.5μM 15μM),L-精氨酸(15μM,1mM 5mM,15mM),β-L-鼠李糖(15μM,1mM,5mM,15mM),β-L-海藻糖(15μM,1mM,5mM,15mM)和L-赖氨酸(15μM,1mM 5mM,15mM)提前加入体系中,进行SDS-PAGE及免疫印迹分析。
5.细胞裂解物中精氨酸鼠李糖基化修饰的检测:分别培养野生型的,缺少EF-P的,缺少糖基转移酶的以及缺少糖基转移底物的Shewanella oneidensis细胞。将细胞裂解液进行SDS-PAGE及免疫印迹分析。实验结果见图6、图7、图8。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员,在不脱离本发明方法的前提下,还可以做出若干改进和补充,这些改进和补充也应视为本发明的保护范围。
Claims (10)
1.一种含有精氨酸鼠李糖基化修饰的糖肽,其特征在于,所述糖肽结构如下所示:
所述含有精氨酸鼠李糖基化修饰的糖肽的制备方法包括如下步骤:
a)化合物2在乙酰氯中反应得到化合物3;
b)化合物3在硫氰化钾与四丁基碘化铵的作用下回流得到化合物4;
c)化合物4在氨气作用下氨化得到化合物5;
d)化合物5在碘乙烷、二碳酸二叔丁酯、三乙胺的作用下分两步反应得到化合物6;
e)以2-氯三苯甲基树脂作为载体,Fmoc-Orn(Alloc)-OH作为糖基化的启动氨基酸,采用基于9-芴甲氧羰基的固相肽合成方法制备化合物7;
f)化合物7与化合物6在硝酸银催化下反应得到化合物8;
g)化合物8在5%的水合肼作用脱去乙酰基,得到目标化合物1;
2.如权利要求1所述的含有精氨酸鼠李糖基化修饰的糖肽的制备方法,其特征在于,包括如下步骤:
a)化合物2在乙酰氯中反应得到化合物3;
b)化合物3在硫氰化钾与四丁基碘化铵的作用下回流得到化合物4;
c)化合物4在氨气作用下氨化得到化合物5;
d)化合物5在碘乙烷、二碳酸二叔丁酯、三乙胺的作用下分两步反应得到化合物6;
e)以2-氯三苯甲基树脂作为载体,Fmoc-Orn(Alloc)-OH作为糖基化的启动氨基酸,采用基于9-芴甲氧羰基的固相肽合成方法制备化合物7;
f)化合物7与化合物6在硝酸银催化下反应得到化合物8;
g)化合物8在5%的水合肼作用脱去乙酰基,得到目标化合物1;
3.由权利要求1所述含有精氨酸鼠李糖基化修饰的糖肽与载体蛋白交联的化合物。
4.根据权利要求3所述的化合物,其特征在于,所述的载体蛋白为BSA,所述的交联为采用Sulfo-GMBS作为交联剂将载体蛋白BSA与糖肽N-端的巯基交联。
5.特异性识别权利要求1所述含有精氨酸鼠李糖基化修饰的糖肽的抗血清或多克隆抗体,其特征在于,以权利要求1所述含有精氨酸鼠李糖基化修饰的糖肽作为半抗原,将该半抗原与载体蛋白偶联获得抗原,再将该抗原经过动物免疫获得抗血清,将抗血清纯化获得多克隆抗体;所述的载体蛋白为BSA,所述的交联为采用Sulfo-GMBS作为交联剂将载体蛋白BSA与糖肽N-端的巯基交联。
6.权利要求5所述多克隆抗体的纯化方法,其特征在于,包括如下步骤:
a)Protein A纯化:纯化系统:AKTApurifier 10,Amersham Biosciences;填料:Protein ASepharose 4Fast Flow;平衡缓冲液:20mM PBS,0.15M NaCl,pH7.2;洗脱缓冲液:0.1M glycine-HCl,pH2.8;上样流速:1ml/min,流程:平衡柱子→上样→再平衡→洗脱,洗脱后样品用1M Tris-HCl,pH=9调pH至中性,PBS透析过夜;
b)特异性纯化:纯化系统:AKTApurifier 10,Amersham Biosciences;填料:BSASepharose 4Fast Flow&CL5(SH)-BSASepharose 4Fast Flow;平衡缓冲液:20mM PBS,0.15M NaCl,pH7.2;洗脱缓冲液:0.1M glycine-HCl,pH=2.8;上样流速:1ml/min;流程:平衡柱子→上样→收集流穿→再上样→收集流穿,洗脱后样品用1M Tris-HCl,pH=9调pH至中性。
7.权利要求5所述的多克隆抗体在检测EF-P的精氨酸鼠李糖修饰上的应用。
8.权利要求5所述的多克隆抗体在检测其它精氨酸鼠李糖基化修饰的蛋白中的应用。
9.权利要求5所述的多克隆抗体在制备抗体类抗生素中的应用。
10.权利要求5所述的多克隆抗体在小分子抗生素筛选中的应用。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201510891290.8A CN105524145B (zh) | 2015-12-07 | 2015-12-07 | 一种高效识别蛋白精氨酸鼠李糖基化的多克隆抗体及其制备方法 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201510891290.8A CN105524145B (zh) | 2015-12-07 | 2015-12-07 | 一种高效识别蛋白精氨酸鼠李糖基化的多克隆抗体及其制备方法 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN105524145A CN105524145A (zh) | 2016-04-27 |
CN105524145B true CN105524145B (zh) | 2019-10-29 |
Family
ID=55766705
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201510891290.8A Active CN105524145B (zh) | 2015-12-07 | 2015-12-07 | 一种高效识别蛋白精氨酸鼠李糖基化的多克隆抗体及其制备方法 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN105524145B (zh) |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109265520A (zh) * | 2018-09-21 | 2019-01-25 | 中国人民解放军第二军医大学 | 一种高效识别蛋白丝氨酸庚糖基化的多克隆抗体、制备方法及其应用 |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101560246A (zh) * | 2009-03-11 | 2009-10-21 | 海南四环心脑血管药物研究院有限公司 | 胸腺五肽衍生物及其制备方法以及该衍生物及其含该衍生物的组合物在制备药物中的应用 |
-
2015
- 2015-12-07 CN CN201510891290.8A patent/CN105524145B/zh active Active
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101560246A (zh) * | 2009-03-11 | 2009-10-21 | 海南四环心脑血管药物研究院有限公司 | 胸腺五肽衍生物及其制备方法以及该衍生物及其含该衍生物的组合物在制备药物中的应用 |
Non-Patent Citations (2)
Title |
---|
Arginine-rhamnosylation as new strategy to activate translation elongation factor P;Lassak J等;《Nature Chemical Biology》;20150430;第266-274页 * |
Synthesis of and Specific Antibody Generation for Glycopeptides with Arginine N-GlcNAcylation;Pan等;《Angewandte Chemie-International Edition》;20141222;第53卷(第52期);第14518-14520页 * |
Also Published As
Publication number | Publication date |
---|---|
CN105524145A (zh) | 2016-04-27 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US10227383B2 (en) | Specific modification of antibody with IgG-binding peptide | |
US20210147512A1 (en) | IgG-BINDING PEPTIDE, AND SPECIFIC MODIFICATION OF ANTIBODY WITH SAID PEPTIDE | |
AU2015254257B2 (en) | Anti-MUC1 antibody or antigen-binding fragment of same, and use thereof | |
JP6077858B2 (ja) | 抗疎水性ペプチド抗体を得るための抗原調製方法 | |
CN111349617A (zh) | 一种分泌抗Tau或pTau-181/231/396单克隆抗体杂交瘤细胞株及其应用 | |
JP5612247B2 (ja) | Cd166に対するモノクローナル抗体およびその産生方法 | |
CN108137651A (zh) | 环肽、亲和层析载体、标记抗体、抗体药物复合物及药物制剂 | |
EP3865515A1 (en) | Solid-phase support including igg-binding peptide, and igg separation method | |
CN105524145B (zh) | 一种高效识别蛋白精氨酸鼠李糖基化的多克隆抗体及其制备方法 | |
WO2016052073A1 (ja) | 抗体結合性ポリペプチド、抗体結合性融合ポリペプチドおよび吸着材料 | |
CN109790202A (zh) | 环肽、亲和层析载体、标记抗体、抗体药物复合体及药物制剂 | |
EP2614371B1 (en) | Method for labeling of compounds | |
Ayuso et al. | Isolation by mAb based affinity chromatography of two Par j I isoallergens. Comparison of their physicochemical, immunochemical and allergenic properties | |
US20020006629A1 (en) | Affinity matrix bearing tumor-associated carbohydrate-or glycopeptide-based antigens and uses thereof | |
Hoschützky et al. | Isolation and characterization of the non-fimbrial adhesin NFA-4 from uropathogenic Escherichia coli O7: K98: H6 | |
CN109897090A (zh) | 一种褐飞虱Vg多肽及其多抗制备方法 | |
KR20090058327A (ko) | 마이코플라즈마 뉴모니아성 폐렴 진단을 위한 재조합단백질 및 이를 이용한 진단 키트 | |
WO2013007667A1 (en) | An antibody specifically binding to neuropilin-1 | |
CN109541199A (zh) | 一种快速检测牛支原体的免疫胶体金试纸及其制备方法 | |
CN104974245B (zh) | 一种褐飞虱VgR多肽及其多克隆抗体制备方法 | |
CN108148124A (zh) | 一种人hnrpa0多肽及其抗体制备方法 | |
CN109265520A (zh) | 一种高效识别蛋白丝氨酸庚糖基化的多克隆抗体、制备方法及其应用 | |
CN109796527A (zh) | 一种红麻线粒体蛋白cox3抗原多肽及制备多克隆抗体的方法和应用 | |
CN101928332A (zh) | 一种人hnrpa0多肽及其抗体制备方法 | |
CN105859838B (zh) | 一种大肠杆菌O157:H7蛋白Ivy多肽、抗Ivy多克隆抗体及其应用 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | Publication | ||
PB01 | Publication | ||
C10 | Entry into substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |