CN106478822B - 一种黄鳝醛酮还原酶多克隆抗体的制备方法 - Google Patents
一种黄鳝醛酮还原酶多克隆抗体的制备方法 Download PDFInfo
- Publication number
- CN106478822B CN106478822B CN201611152038.6A CN201611152038A CN106478822B CN 106478822 B CN106478822 B CN 106478822B CN 201611152038 A CN201611152038 A CN 201611152038A CN 106478822 B CN106478822 B CN 106478822B
- Authority
- CN
- China
- Prior art keywords
- swamp eel
- eakr
- polyclonal antibody
- preparation
- gene
- 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
- 241000646357 Monopterus cuchia Species 0.000 title claims abstract description 103
- 238000002360 preparation method Methods 0.000 title claims abstract description 28
- 150000001299 aldehydes Chemical class 0.000 title claims abstract description 23
- 108010031132 Alcohol Oxidoreductases Proteins 0.000 title claims abstract description 21
- 102000005751 Alcohol Oxidoreductases Human genes 0.000 title claims abstract description 20
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 55
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 27
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 claims abstract description 24
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 claims abstract description 24
- 230000014509 gene expression Effects 0.000 claims abstract description 15
- 238000003259 recombinant expression Methods 0.000 claims abstract description 13
- 238000001262 western blot Methods 0.000 claims abstract description 12
- 238000001514 detection method Methods 0.000 claims abstract description 10
- 230000003053 immunization Effects 0.000 claims abstract description 9
- 238000002649 immunization Methods 0.000 claims abstract description 8
- 230000001939 inductive effect Effects 0.000 claims abstract description 8
- 239000013598 vector Substances 0.000 claims abstract description 4
- 210000002966 serum Anatomy 0.000 claims description 25
- 239000002299 complementary DNA Substances 0.000 claims description 23
- 238000006243 chemical reaction Methods 0.000 claims description 19
- 238000012408 PCR amplification Methods 0.000 claims description 12
- 241000283973 Oryctolagus cuniculus Species 0.000 claims description 10
- 238000001962 electrophoresis Methods 0.000 claims description 10
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 claims description 10
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 claims description 10
- 230000029087 digestion Effects 0.000 claims description 8
- 241000588724 Escherichia coli Species 0.000 claims description 7
- 239000000243 solution Substances 0.000 claims description 7
- 241000894006 Bacteria Species 0.000 claims description 6
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical class 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 6
- 229930027917 kanamycin Natural products 0.000 claims description 6
- 239000000047 product Substances 0.000 claims description 6
- 239000002671 adjuvant Substances 0.000 claims description 5
- 238000005119 centrifugation Methods 0.000 claims description 5
- 239000012634 fragment Substances 0.000 claims description 5
- 238000011587 new zealand white rabbit Methods 0.000 claims description 5
- 239000002773 nucleotide Substances 0.000 claims description 5
- 125000003729 nucleotide group Chemical group 0.000 claims description 5
- 230000001376 precipitating effect Effects 0.000 claims description 5
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 claims description 4
- 239000000427 antigen Substances 0.000 claims description 4
- 102000036639 antigens Human genes 0.000 claims description 4
- 108091007433 antigens Proteins 0.000 claims description 4
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 claims description 4
- 238000002347 injection Methods 0.000 claims description 4
- 239000007924 injection Substances 0.000 claims description 4
- 238000011144 upstream manufacturing Methods 0.000 claims description 4
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 3
- 102100034343 Integrase Human genes 0.000 claims description 3
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 claims description 3
- 210000004369 blood Anatomy 0.000 claims description 3
- 239000008280 blood Substances 0.000 claims description 3
- 238000013461 design Methods 0.000 claims description 3
- 238000000502 dialysis Methods 0.000 claims description 3
- 239000001963 growth medium Substances 0.000 claims description 3
- 239000012160 loading buffer Substances 0.000 claims description 3
- 239000012139 lysis buffer Substances 0.000 claims description 3
- 238000000926 separation method Methods 0.000 claims description 3
- 239000006228 supernatant Substances 0.000 claims description 3
- 238000010828 elution Methods 0.000 claims description 2
- 239000006167 equilibration buffer Substances 0.000 claims description 2
- 239000013604 expression vector Substances 0.000 claims description 2
- 229960000789 guanidine hydrochloride Drugs 0.000 claims description 2
- 150000002460 imidazoles Chemical class 0.000 claims description 2
- 230000006698 induction Effects 0.000 claims description 2
- 239000002054 inoculum Substances 0.000 claims description 2
- 238000011068 loading method Methods 0.000 claims description 2
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 claims description 2
- 238000011084 recovery Methods 0.000 claims description 2
- 241000305071 Enterobacterales Species 0.000 claims 1
- 125000003275 alpha amino acid group Chemical group 0.000 claims 1
- 239000003795 chemical substances by application Substances 0.000 claims 1
- 238000002525 ultrasonication Methods 0.000 claims 1
- 238000000034 method Methods 0.000 abstract description 18
- 238000011160 research Methods 0.000 abstract description 5
- 241001597062 Channa argus Species 0.000 abstract description 4
- 241000252230 Ctenopharyngodon idella Species 0.000 abstract description 4
- 241001275890 Megalobrama amblycephala Species 0.000 abstract description 4
- 238000001042 affinity chromatography Methods 0.000 abstract description 4
- 238000004458 analytical method Methods 0.000 abstract description 4
- 210000001061 forehead Anatomy 0.000 abstract description 4
- 238000010353 genetic engineering Methods 0.000 abstract description 4
- 238000004043 dyeing Methods 0.000 abstract description 3
- 238000002474 experimental method Methods 0.000 abstract description 3
- 238000003119 immunoblot Methods 0.000 abstract description 3
- 238000010166 immunofluorescence Methods 0.000 abstract description 3
- 238000003364 immunohistochemistry Methods 0.000 abstract 1
- 235000019688 fish Nutrition 0.000 description 14
- 241000251468 Actinopterygii Species 0.000 description 13
- 210000004185 liver Anatomy 0.000 description 12
- 239000013642 negative control Substances 0.000 description 11
- 238000010790 dilution Methods 0.000 description 9
- 239000012895 dilution Substances 0.000 description 9
- 241000283707 Capra Species 0.000 description 8
- 210000004923 pancreatic tissue Anatomy 0.000 description 8
- 210000000952 spleen Anatomy 0.000 description 8
- 238000011534 incubation Methods 0.000 description 7
- 210000000813 small intestine Anatomy 0.000 description 7
- 230000008901 benefit Effects 0.000 description 6
- 239000000872 buffer Substances 0.000 description 6
- 239000000203 mixture Substances 0.000 description 6
- 239000013612 plasmid Substances 0.000 description 6
- 230000008569 process Effects 0.000 description 6
- 210000001519 tissue Anatomy 0.000 description 6
- 238000012360 testing method Methods 0.000 description 5
- 241001465754 Metazoa Species 0.000 description 4
- 230000008859 change Effects 0.000 description 4
- 238000000751 protein extraction Methods 0.000 description 4
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 241000158704 Monopterus albus Species 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 235000013601 eggs Nutrition 0.000 description 3
- 238000002991 immunohistochemical analysis Methods 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 210000003205 muscle Anatomy 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 230000006798 recombination Effects 0.000 description 3
- 238000005215 recombination Methods 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 238000011895 specific detection Methods 0.000 description 3
- 238000004448 titration Methods 0.000 description 3
- WZUVPPKBWHMQCE-XJKSGUPXSA-N (+)-haematoxylin Chemical compound C12=CC(O)=C(O)C=C2C[C@]2(O)[C@H]1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-XJKSGUPXSA-N 0.000 description 2
- 241000252229 Carassius auratus Species 0.000 description 2
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 2
- 208000002249 Diabetes Complications Diseases 0.000 description 2
- 206010012655 Diabetic complications Diseases 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 101150092780 GSP1 gene Proteins 0.000 description 2
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Natural products C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 description 2
- DZQMXBALGUHGJT-GUBZILKMSA-N Leu-Glu-Ala Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(O)=O DZQMXBALGUHGJT-GUBZILKMSA-N 0.000 description 2
- IEWBEPKLKUXQBU-VOAKCMCISA-N Leu-Leu-Thr Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O IEWBEPKLKUXQBU-VOAKCMCISA-N 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 2
- 241000276701 Oreochromis mossambicus Species 0.000 description 2
- 102000003992 Peroxidases Human genes 0.000 description 2
- MUJQWSAWLLRJCE-KATARQTJSA-N Ser-Leu-Thr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O MUJQWSAWLLRJCE-KATARQTJSA-N 0.000 description 2
- 239000006180 TBST buffer Substances 0.000 description 2
- 150000001413 amino acids Chemical group 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 238000009835 boiling Methods 0.000 description 2
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 2
- 239000005515 coenzyme Substances 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 239000012141 concentrate Substances 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 238000001784 detoxification Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 238000011010 flushing procedure Methods 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- 150000002576 ketones Chemical class 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 239000012188 paraffin wax Substances 0.000 description 2
- 108040007629 peroxidase activity proteins Proteins 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 239000013049 sediment Substances 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 239000001993 wax Substances 0.000 description 2
- 238000004383 yellowing Methods 0.000 description 2
- COEXAQSTZUWMRI-STQMWFEESA-N (2s)-1-[2-[[(2s)-2-amino-3-(4-hydroxyphenyl)propanoyl]amino]acetyl]pyrrolidine-2-carboxylic acid Chemical compound C([C@H](N)C(=O)NCC(=O)N1[C@@H](CCC1)C(O)=O)C1=CC=C(O)C=C1 COEXAQSTZUWMRI-STQMWFEESA-N 0.000 description 1
- RXMWXENJQAINCC-DMTCNVIQSA-N 2,5-didehydro-D-gluconic acid Chemical compound OCC(=O)[C@@H](O)[C@H](O)C(=O)C(O)=O RXMWXENJQAINCC-DMTCNVIQSA-N 0.000 description 1
- RXMWXENJQAINCC-UHFFFAOYSA-N 2,5-diketo-D-gluconic acid Natural products OCC(=O)C(O)C(O)C(=O)C(O)=O RXMWXENJQAINCC-UHFFFAOYSA-N 0.000 description 1
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 description 1
- 101150061567 AKR1A1 gene Proteins 0.000 description 1
- 229930195730 Aflatoxin Natural products 0.000 description 1
- GWFSQQNGMPGBEF-GHCJXIJMSA-N Ala-Asp-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](C)N GWFSQQNGMPGBEF-GHCJXIJMSA-N 0.000 description 1
- WKOBSJOZRJJVRZ-FXQIFTODSA-N Ala-Glu-Glu Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O WKOBSJOZRJJVRZ-FXQIFTODSA-N 0.000 description 1
- VBRDBGCROKWTPV-XHNCKOQMSA-N Ala-Glu-Pro Chemical compound C[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N1CCC[C@@H]1C(=O)O)N VBRDBGCROKWTPV-XHNCKOQMSA-N 0.000 description 1
- GSHKMNKPMLXSQW-KBIXCLLPSA-N Ala-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](C)N GSHKMNKPMLXSQW-KBIXCLLPSA-N 0.000 description 1
- ZCUFMRIQCPNOHZ-NRPADANISA-N Ala-Val-Gln Chemical compound C[C@@H](C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N ZCUFMRIQCPNOHZ-NRPADANISA-N 0.000 description 1
- 108010053754 Aldehyde reductase Proteins 0.000 description 1
- 108010084469 Aldo-Keto Reductases Proteins 0.000 description 1
- 102000005602 Aldo-Keto Reductases Human genes 0.000 description 1
- 102100026448 Aldo-keto reductase family 1 member A1 Human genes 0.000 description 1
- 102100027265 Aldo-keto reductase family 1 member B1 Human genes 0.000 description 1
- NKBQZKVMKJJDLX-SRVKXCTJSA-N Arg-Glu-Leu Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O NKBQZKVMKJJDLX-SRVKXCTJSA-N 0.000 description 1
- PAPSMOYMQDWIOR-AVGNSLFASA-N Arg-Lys-Val Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(O)=O PAPSMOYMQDWIOR-AVGNSLFASA-N 0.000 description 1
- BSYKSCBTTQKOJG-GUBZILKMSA-N Arg-Pro-Ala Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C)C(O)=O BSYKSCBTTQKOJG-GUBZILKMSA-N 0.000 description 1
- VENMDXUVHSKEIN-GUBZILKMSA-N Arg-Ser-Arg Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O VENMDXUVHSKEIN-GUBZILKMSA-N 0.000 description 1
- HZPSDHRYYIORKR-WHFBIAKZSA-N Asn-Ala-Gly Chemical compound OC(=O)CNC(=O)[C@H](C)NC(=O)[C@@H](N)CC(N)=O HZPSDHRYYIORKR-WHFBIAKZSA-N 0.000 description 1
- SRUUBQBAVNQZGJ-LAEOZQHASA-N Asn-Gln-Val Chemical compound CC(C)[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CC(=O)N)N SRUUBQBAVNQZGJ-LAEOZQHASA-N 0.000 description 1
- LSJQOMAZIKQMTJ-SRVKXCTJSA-N Asn-Phe-Asp Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(O)=O)C(O)=O LSJQOMAZIKQMTJ-SRVKXCTJSA-N 0.000 description 1
- UGXYFDQFLVCDFC-CIUDSAMLSA-N Asn-Ser-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O UGXYFDQFLVCDFC-CIUDSAMLSA-N 0.000 description 1
- KRXIWXCXOARFNT-ZLUOBGJFSA-N Asp-Ala-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC(O)=O KRXIWXCXOARFNT-ZLUOBGJFSA-N 0.000 description 1
- RRKCPMGSRIDLNC-AVGNSLFASA-N Asp-Glu-Tyr Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O RRKCPMGSRIDLNC-AVGNSLFASA-N 0.000 description 1
- 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 1
- KFAFUJMGHVVYRC-DCAQKATOSA-N Asp-Leu-Met Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(O)=O KFAFUJMGHVVYRC-DCAQKATOSA-N 0.000 description 1
- CZIVKMOEXPILDK-SRVKXCTJSA-N Asp-Tyr-Ser Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CO)C(O)=O CZIVKMOEXPILDK-SRVKXCTJSA-N 0.000 description 1
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 108010061982 DNA Ligases Proteins 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 102000002322 Egg Proteins Human genes 0.000 description 1
- 108010000912 Egg Proteins Proteins 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- KQOPMGBHNQBCEL-HVTMNAMFSA-N Gln-His-Ile Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O KQOPMGBHNQBCEL-HVTMNAMFSA-N 0.000 description 1
- FITIQFSXXBKFFM-NRPADANISA-N Gln-Val-Ser Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(O)=O FITIQFSXXBKFFM-NRPADANISA-N 0.000 description 1
- GCYFUZJHAXJKKE-KKUMJFAQSA-N Glu-Arg-Tyr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O GCYFUZJHAXJKKE-KKUMJFAQSA-N 0.000 description 1
- RFDHKPSHTXZKLL-IHRRRGAJSA-N Glu-Gln-Phe Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CCC(=O)O)N RFDHKPSHTXZKLL-IHRRRGAJSA-N 0.000 description 1
- AUTNXSQEVVHSJK-YVNDNENWSA-N Glu-Glu-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)CCC(O)=O AUTNXSQEVVHSJK-YVNDNENWSA-N 0.000 description 1
- IOUQWHIEQYQVFD-JYJNAYRXSA-N Glu-Leu-Tyr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O IOUQWHIEQYQVFD-JYJNAYRXSA-N 0.000 description 1
- JRDYDYXZKFNNRQ-XPUUQOCRSA-N Gly-Ala-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)CN JRDYDYXZKFNNRQ-XPUUQOCRSA-N 0.000 description 1
- SUDUYJOBLHQAMI-WHFBIAKZSA-N Gly-Asp-Cys Chemical compound NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CS)C(O)=O SUDUYJOBLHQAMI-WHFBIAKZSA-N 0.000 description 1
- LCNXZQROPKFGQK-WHFBIAKZSA-N Gly-Asp-Ser Chemical compound NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(O)=O LCNXZQROPKFGQK-WHFBIAKZSA-N 0.000 description 1
- SXJHOPPTOJACOA-QXEWZRGKSA-N Gly-Ile-Arg Chemical compound NCC(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@H](C(O)=O)CCCN=C(N)N SXJHOPPTOJACOA-QXEWZRGKSA-N 0.000 description 1
- LRQXRHGQEVWGPV-NHCYSSNCSA-N Gly-Leu-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)CN LRQXRHGQEVWGPV-NHCYSSNCSA-N 0.000 description 1
- IUKIDFVOUHZRAK-QWRGUYRKSA-N Gly-Lys-His Chemical compound NCCCC[C@H](NC(=O)CN)C(=O)N[C@H](C(O)=O)CC1=CN=CN1 IUKIDFVOUHZRAK-QWRGUYRKSA-N 0.000 description 1
- TTYVAUJGNMVTRN-GJZGRUSLSA-N Gly-Met-Trp Chemical compound CSCC[C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)O)NC(=O)CN TTYVAUJGNMVTRN-GJZGRUSLSA-N 0.000 description 1
- OCPPBNKYGYSLOE-IUCAKERBSA-N Gly-Pro-Met Chemical compound CSCC[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)CN OCPPBNKYGYSLOE-IUCAKERBSA-N 0.000 description 1
- GWCJMBNBFYBQCV-XPUUQOCRSA-N Gly-Val-Ala Chemical compound NCC(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C)C(O)=O GWCJMBNBFYBQCV-XPUUQOCRSA-N 0.000 description 1
- BNBQSLZMHBFEIV-TUSQITKMSA-N His-Trp-Trp Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(O)=O)C1=CN=CN1 BNBQSLZMHBFEIV-TUSQITKMSA-N 0.000 description 1
- 108010033040 Histones Proteins 0.000 description 1
- 101000718007 Homo sapiens Aldo-keto reductase family 1 member A1 Proteins 0.000 description 1
- 101000911390 Homo sapiens Coagulation factor VIII Proteins 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 102000011145 Hydroxysteroid Dehydrogenases Human genes 0.000 description 1
- 108010062875 Hydroxysteroid Dehydrogenases Proteins 0.000 description 1
- UDLAWRKOVFDKFL-PEFMBERDSA-N Ile-Asp-Gln Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N UDLAWRKOVFDKFL-PEFMBERDSA-N 0.000 description 1
- GYAFMRQGWHXMII-IUKAMOBKSA-N Ile-Asp-Thr Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H]([C@@H](C)O)C(=O)O)N GYAFMRQGWHXMII-IUKAMOBKSA-N 0.000 description 1
- PXKACEXYLPBMAD-JBDRJPRFSA-N Ile-Ser-Ser Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)O)N PXKACEXYLPBMAD-JBDRJPRFSA-N 0.000 description 1
- 108010009384 L-Iditol 2-Dehydrogenase Proteins 0.000 description 1
- CZCSUZMIRKFFFA-CIUDSAMLSA-N Leu-Ala-Asn Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(N)=O)C(O)=O CZCSUZMIRKFFFA-CIUDSAMLSA-N 0.000 description 1
- WNGVUZWBXZKQES-YUMQZZPRSA-N Leu-Ala-Gly Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C)C(=O)NCC(O)=O WNGVUZWBXZKQES-YUMQZZPRSA-N 0.000 description 1
- FGNQZXKVAZIMCI-CIUDSAMLSA-N Leu-Asp-Cys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CS)C(=O)O)N FGNQZXKVAZIMCI-CIUDSAMLSA-N 0.000 description 1
- 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 1
- VBZOAGIPCULURB-QWRGUYRKSA-N Leu-Gly-His Chemical compound CC(C)C[C@@H](C(=O)NCC(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N VBZOAGIPCULURB-QWRGUYRKSA-N 0.000 description 1
- APFJUBGRZGMQFF-QWRGUYRKSA-N Leu-Gly-Lys Chemical compound CC(C)C[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCCCN APFJUBGRZGMQFF-QWRGUYRKSA-N 0.000 description 1
- OYQUOLRTJHWVSQ-SRVKXCTJSA-N Leu-His-Ser Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CO)C(O)=O OYQUOLRTJHWVSQ-SRVKXCTJSA-N 0.000 description 1
- ZAVCJRJOQKIOJW-KKUMJFAQSA-N Leu-Phe-Asp Chemical compound CC(C)C[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](CC(O)=O)C(O)=O)CC1=CC=CC=C1 ZAVCJRJOQKIOJW-KKUMJFAQSA-N 0.000 description 1
- GOFJOGXGMPHOGL-DCAQKATOSA-N Leu-Ser-Met Chemical compound CSCC[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(C)C GOFJOGXGMPHOGL-DCAQKATOSA-N 0.000 description 1
- SIGZKCWZEBFNAK-QAETUUGQSA-N Leu-Ser-Ser-Tyr Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 SIGZKCWZEBFNAK-QAETUUGQSA-N 0.000 description 1
- GZRABTMNWJXFMH-UVOCVTCTSA-N Leu-Thr-Thr Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O GZRABTMNWJXFMH-UVOCVTCTSA-N 0.000 description 1
- QESXLSQLQHHTIX-RHYQMDGZSA-N Leu-Val-Thr Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O QESXLSQLQHHTIX-RHYQMDGZSA-N 0.000 description 1
- GGAPIOORBXHMNY-ULQDDVLXSA-N Lys-Arg-Tyr Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CCCCN)N)O GGAPIOORBXHMNY-ULQDDVLXSA-N 0.000 description 1
- RBEATVHTWHTHTJ-KKUMJFAQSA-N Lys-Leu-Lys Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(O)=O RBEATVHTWHTHTJ-KKUMJFAQSA-N 0.000 description 1
- KJIXWRWPOCKYLD-IHRRRGAJSA-N Lys-Lys-Met Chemical compound CSCC[C@@H](C(=O)O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCCCN)N KJIXWRWPOCKYLD-IHRRRGAJSA-N 0.000 description 1
- VVURYEVJJTXWNE-ULQDDVLXSA-N Lys-Tyr-Val Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](C(C)C)C(O)=O VVURYEVJJTXWNE-ULQDDVLXSA-N 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- JYCQGAGDJQYEDB-GUBZILKMSA-N Met-Gln-Gln Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O JYCQGAGDJQYEDB-GUBZILKMSA-N 0.000 description 1
- BJPQKNHZHUCQNQ-SRVKXCTJSA-N Met-Pro-Val Chemical compound CC(C)[C@@H](C(=O)O)NC(=O)[C@@H]1CCCN1C(=O)[C@H](CCSC)N BJPQKNHZHUCQNQ-SRVKXCTJSA-N 0.000 description 1
- AJHCSUXXECOXOY-UHFFFAOYSA-N N-glycyl-L-tryptophan Natural products C1=CC=C2C(CC(NC(=O)CN)C(O)=O)=CNC2=C1 AJHCSUXXECOXOY-UHFFFAOYSA-N 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 108010033276 Peptide Fragments Proteins 0.000 description 1
- 102000007079 Peptide Fragments Human genes 0.000 description 1
- CDNPIRSCAFMMBE-SRVKXCTJSA-N Phe-Asn-Ser Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(O)=O CDNPIRSCAFMMBE-SRVKXCTJSA-N 0.000 description 1
- IQXOZIDWLZYYAW-IHRRRGAJSA-N Phe-Asp-Met Chemical compound CSCC[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)N IQXOZIDWLZYYAW-IHRRRGAJSA-N 0.000 description 1
- SXJGROGVINAYSH-AVGNSLFASA-N Phe-Gln-Asp Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CC(=O)O)C(=O)O)N SXJGROGVINAYSH-AVGNSLFASA-N 0.000 description 1
- YYKZDTVQHTUKDW-RYUDHWBXSA-N Phe-Gly-Gln Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)NCC(=O)N[C@@H](CCC(=O)N)C(=O)O)N YYKZDTVQHTUKDW-RYUDHWBXSA-N 0.000 description 1
- SKICPQLTOXGWGO-GARJFASQSA-N Pro-Gln-Pro Chemical compound C1C[C@H](NC1)C(=O)N[C@@H](CCC(=O)N)C(=O)N2CCC[C@@H]2C(=O)O SKICPQLTOXGWGO-GARJFASQSA-N 0.000 description 1
- HAAQQNHQZBOWFO-LURJTMIESA-N Pro-Gly-Gly Chemical compound OC(=O)CNC(=O)CNC(=O)[C@@H]1CCCN1 HAAQQNHQZBOWFO-LURJTMIESA-N 0.000 description 1
- 238000012181 QIAquick gel extraction kit Methods 0.000 description 1
- NCYCYZXNIZJOKI-OVSJKPMPSA-N Retinaldehyde Chemical compound O=C\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C NCYCYZXNIZJOKI-OVSJKPMPSA-N 0.000 description 1
- OBXVZEAMXFSGPU-FXQIFTODSA-N Ser-Asn-Arg Chemical compound C(C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CO)N)CN=C(N)N OBXVZEAMXFSGPU-FXQIFTODSA-N 0.000 description 1
- 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 1
- 102100026974 Sorbitol dehydrogenase Human genes 0.000 description 1
- DWYAUVCQDTZIJI-VZFHVOOUSA-N Thr-Ala-Ser Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O DWYAUVCQDTZIJI-VZFHVOOUSA-N 0.000 description 1
- XVNZSJIKGJLQLH-RCWTZXSCSA-N Thr-Arg-Met Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCSC)C(=O)O)N)O XVNZSJIKGJLQLH-RCWTZXSCSA-N 0.000 description 1
- QILPDQCTQZDHFM-HJGDQZAQSA-N Thr-Gln-Arg Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O QILPDQCTQZDHFM-HJGDQZAQSA-N 0.000 description 1
- 102100036502 Trans-1,2-dihydrobenzene-1,2-diol dehydrogenase Human genes 0.000 description 1
- 108010039246 Trans-1,2-dihydrobenzene-1,2-diol dehydrogenase Proteins 0.000 description 1
- JVTHMUDOKPQBOT-NSHDSACASA-N Trp-Gly-Gly Chemical compound C1=CC=C2C(C[C@H]([NH3+])C(=O)NCC(=O)NCC([O-])=O)=CNC2=C1 JVTHMUDOKPQBOT-NSHDSACASA-N 0.000 description 1
- BOBZBMOTRORUPT-XIRDDKMYSA-N Trp-Ser-Leu Chemical compound C1=CC=C2C(C[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(O)=O)=CNC2=C1 BOBZBMOTRORUPT-XIRDDKMYSA-N 0.000 description 1
- QAYSODICXVZUIA-WLTAIBSBSA-N Tyr-Gly-Thr Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)NCC(=O)N[C@@H]([C@@H](C)O)C(O)=O QAYSODICXVZUIA-WLTAIBSBSA-N 0.000 description 1
- TYFLVOUZHQUBGM-IHRRRGAJSA-N Tyr-Ser-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 TYFLVOUZHQUBGM-IHRRRGAJSA-N 0.000 description 1
- YODDULVCGFQRFZ-ZKWXMUAHSA-N Val-Asp-Ser Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(O)=O YODDULVCGFQRFZ-ZKWXMUAHSA-N 0.000 description 1
- JXGWQYWDUOWQHA-DZKIICNBSA-N Val-Gln-Phe Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)N JXGWQYWDUOWQHA-DZKIICNBSA-N 0.000 description 1
- ZXAGTABZUOMUDO-GVXVVHGQSA-N Val-Glu-Lys Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CCCCN)C(=O)O)N ZXAGTABZUOMUDO-GVXVVHGQSA-N 0.000 description 1
- FEXILLGKGGTLRI-NHCYSSNCSA-N Val-Leu-Asn Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](C(C)C)N FEXILLGKGGTLRI-NHCYSSNCSA-N 0.000 description 1
- IEBGHUMBJXIXHM-AVGNSLFASA-N Val-Lys-Met Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCSC)C(=O)O)N IEBGHUMBJXIXHM-AVGNSLFASA-N 0.000 description 1
- BGXVHVMJZCSOCA-AVGNSLFASA-N Val-Pro-Lys Chemical compound CC(C)[C@@H](C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCCN)C(=O)O)N BGXVHVMJZCSOCA-AVGNSLFASA-N 0.000 description 1
- UQMPYVLTQCGRSK-IFFSRLJSSA-N Val-Thr-Gln Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)O)NC(=O)[C@H](C(C)C)N)O UQMPYVLTQCGRSK-IFFSRLJSSA-N 0.000 description 1
- LCHZBEUVGAVMKS-RHYQMDGZSA-N Val-Thr-Leu Chemical compound CC(C)C[C@H](NC(=O)[C@@H](NC(=O)[C@@H](N)C(C)C)[C@@H](C)O)C(O)=O LCHZBEUVGAVMKS-RHYQMDGZSA-N 0.000 description 1
- WFTKOJGOOUJLJV-VKOGCVSHSA-N Val-Trp-Ile Chemical compound C1=CC=C2C(C[C@@H](C(=O)N[C@@H]([C@@H](C)CC)C([O-])=O)NC(=O)[C@@H]([NH3+])C(C)C)=CNC2=C1 WFTKOJGOOUJLJV-VKOGCVSHSA-N 0.000 description 1
- 239000005409 aflatoxin Substances 0.000 description 1
- 108010076324 alanyl-glycyl-glycine Proteins 0.000 description 1
- 108010087924 alanylproline Proteins 0.000 description 1
- -1 alcohol compound Chemical class 0.000 description 1
- 238000003452 antibody preparation method Methods 0.000 description 1
- 150000001491 aromatic compounds Chemical class 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 230000000711 cancerogenic effect Effects 0.000 description 1
- 150000001728 carbonyl compounds Chemical class 0.000 description 1
- 231100000315 carcinogenic Toxicity 0.000 description 1
- 150000003943 catecholamines Chemical class 0.000 description 1
- 238000005138 cryopreservation Methods 0.000 description 1
- 108010016616 cysteinylglycine Proteins 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 235000014103 egg white Nutrition 0.000 description 1
- 210000000969 egg white Anatomy 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000003925 fat Substances 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 239000013505 freshwater Substances 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 108010026364 glycyl-glycyl-leucine Proteins 0.000 description 1
- 230000002440 hepatic effect Effects 0.000 description 1
- 108010036413 histidylglycine Proteins 0.000 description 1
- 108010025306 histidylleucine Proteins 0.000 description 1
- 108010092114 histidylphenylalanine Proteins 0.000 description 1
- 102000057593 human F8 Human genes 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 108010044374 isoleucyl-tyrosine Proteins 0.000 description 1
- 108010027338 isoleucylcysteine Proteins 0.000 description 1
- 108010057821 leucylproline Proteins 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 108010017391 lysylvaline Proteins 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 108010056582 methionylglutamic acid Proteins 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 229910052759 nickel Inorganic materials 0.000 description 1
- 239000002777 nucleoside Substances 0.000 description 1
- 125000003835 nucleoside group Chemical group 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 108010014614 prolyl-glycyl-proline Proteins 0.000 description 1
- 150000003180 prostaglandins Chemical class 0.000 description 1
- 229940047431 recombinate Drugs 0.000 description 1
- 238000004153 renaturation Methods 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 235000020945 retinal Nutrition 0.000 description 1
- 239000011604 retinal Substances 0.000 description 1
- 108010048397 seryl-lysyl-leucine Proteins 0.000 description 1
- 150000003431 steroids Chemical class 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 108010058119 tryptophyl-glycyl-glycine Proteins 0.000 description 1
- 108010051110 tyrosyl-lysine Proteins 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 108700026220 vif Genes Proteins 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0008—Oxidoreductases (1.) acting on the aldehyde or oxo group of donors (1.2)
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Immunology (AREA)
- Biophysics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
本发明公开了一种黄鳝醛酮还原酶多克隆抗体的制备方法，包括(1)黄鳝Eakr基因的克隆；(2)黄鳝Eakr基因重组表达载体的构建；(3)黄鳝Eakr重组蛋白的诱导表达和纯化；(4)多克隆抗体的制备及Western blot分析。本发明采用分子生物学和基因工程的方法，构建了黄鳝Eakr基因的重组表达载体，诱导表达，亲和层析获得纯化的重组表达蛋白。本发明的方法制备的黄鳝Eakr多克隆抗体可用于免疫组化、免疫印迹实验要求，建立体外免疫分析、研究黄鳝Eakr的功能以及黄鳝免疫荧光染色激光共聚焦显微观察，还可用于草鱼、黄颡、团头鲂、乌鳢和鲫鱼的醛酮还原酶的免疫检测。
Description
技术领域
本发明属于基因工程领域，具体的说，涉及一种黄鳝醛酮还原酶多克隆抗体的制备方法。
背景技术
醛酮还原酶(Aldo-keto reductase，AKR)广泛存在于生物界，是一类依赖于NAD(P)H的分子量在34kDa-37kDa之间的单体还原酶蛋白。
现报道的AKR超家族成员有16个亚家族共190多个成员，包括醛糖还原酶、酮还原酶、羟化类固醇脱氢酶、2,5-二酮基-D-葡萄糖酸还原酶、多元醇脱氢酶、二氢二醇脱氢酶等多种能够代谢机体羰基化合物的酶类。
在结构上，AKR超家族都包含一个(α/β)8的桶状结构、相似的辅酶结合结构域和一个高度保守的Asp，Tyr，Lys，His催化四分体。在功能上，AKR不仅在生物体应对外源性或内源性羰基化合物解毒过程中具有重要作用，还参与了生物体内氧化应激、致癌化合物的降解、糖尿病并发症及肿瘤发生等过程。
通过辅酶NAD(P)H提供还原力，AKR可以将醛酮类化合物通过加氢的方式还原成相对应的醇类化合物，从而降低毒害作用。其底物包括脂肪类醛酮化合物、芳香类化合物、醛糖、儿茶酚胺、甾类、视黄醛、前列腺素、黄曲霉素等。研究发现，AKR除了参与机体的疾病发生过程，还作为重要的防御酶存在。AKR抑制剂在癌症及糖尿病并发症临床治疗等方面具有重要的意义，目前已成为研究的热点。
相比哺乳动物，对于鱼类AKR的研究报道较少。国外有学者研究了苯并芘(BaP)处理罗非鱼后其肝组织AKR1A1基因的表达情况，认为罗非鱼的AKR1A1可能在BaP解毒过程中具有重要的作用。除此之外，其它鱼类的AKR基因及其功能的研究国内外尚无报道。
从黄鳝(Monopterus albus)中克隆到醛酮还原酶(Eakr)基因，对其进行体外表达、多克隆抗体制备及抗体检测的研究，对于进一步探索鱼类醛酮还原酶基因的功能具有重要意义。
发明内容
有鉴于此，有必要提供一种能制备出大量高质量的抗黄鳝Eakr的多克隆抗体的黄鳝Eakr多克隆抗体的制备方法。
一种黄鳝Eakr多克隆抗体的制备方法，包括以下步骤：
步骤(1)黄鳝Eakr基因的克隆：
按照Trizol试剂说明书提取黄鳝总RNA，用M-MLV反转录酶合成cDNA第一链，于-80℃保存；根据GenBank数据库中已报道的AKR基因序列，设计引物，进行PCR扩增,获得黄鳝Eakr基因cDNA片段；根据Smart RACE试剂盒说明书，进行5’RACE-PCR和3’RACE-PCR扩增，获得黄鳝Eakr基因cDNA片段的5’末端和3’末端，拼接获得其全长cDNA序列；黄鳝Eakr基因的核苷酸和氨基酸序列分别如SEQ ID NO：1和SEQ ID NO：2所示；
步骤(2)黄鳝Eakr基因重组表达载体的构建：
根据黄鳝Eakr基因全长cDNA序列，设计一对特异性引物re-ex-F(5’-GCCGAATTCATGCCTGTGGTTCCCAAAG-3’)和re-ex-R(5’-CCGAAGCTTTTAGCTCCTGTACTCATCGC-3’)，分别在上、下游引物添加了EcoR I和Hind III酶切位点；以黄鳝cDNA为模板，re-ex-F和re-ex-R为引物，进行PCR扩增；将PCR产物和pET-28a(+)表达载体进行双酶切，回收纯化，连接转化大肠杆菌DH5α，获得重组表达载体pET-Eakr；
步骤(3)黄鳝Eakr重组蛋白的诱导表达和纯化：
将步骤(2)得到的重组表达载体pET-Eakr转化大肠杆菌BL21(DE3)，在含有50mg/L卡那霉素的LB培养液中培养至OD值为0.6时，加入IPTG进行诱导，培养3h。离心收集菌体，超声破碎，然后加入5×上样缓冲液进行12％SDS-PAGE电泳检测目标蛋白的表达情况；
以1％的接种量将能表达重组蛋白的BL21添加到含有50μg/mL卡那霉素的LB培养基中，大量培养，诱导表达；将诱导好的菌液离心，收集菌体超声波破碎，再离心收集沉淀；将沉淀用平衡缓冲液溶解，收集上清，上样、结合、洗脱、透析，获得纯化的黄鳝Eakr重组蛋白；SDS-PAGE电泳检测纯化后的黄鳝Eakr重组蛋白；
步骤(4)多克隆抗体的制备及Western blot分析：
将步骤(3)纯化的黄鳝pET-Eakr重组蛋白作为抗原，对新西兰大白兔进行8次免疫，按体积比1︰1的比例加入弗氏完全佐剂，采用背部皮下多点注射，免疫剂量为0.2mg；加强免疫选用弗氏不完全佐剂；末次免疫10天后，进行全血分离，免疫血清储存于-80℃备用。提取经IPTG诱导和未诱导的pET-Eakr重组菌的融合蛋白进行Western blot，检测抗体的特异性；
为进一步分析黄鳝pET-Eakr多克隆抗体的特异性，利用动物组织总蛋白提取试剂盒提取黄鳝肝胰组织、小肠、肌肉和脾脏4种组织的总蛋白；取50μg各组织总蛋白进行SDS-PAGE电泳，然后将蛋白转至NC膜上。用制备的抗血清进行1：500倍稀释后作为一抗，用HRP标记的羊抗兔IgG为二抗进行多抗的特异性检测；
步骤(5)免疫组化分析：
采用石蜡包埋法包埋黄鳝的小肠、肝胰组织和脾脏,以6μm厚度进行切片；常规脱蜡后，用50μL的H2O2(3％)于37℃孵育10min，阻断内源过氧化物酶；将切片放入柠檬酸盐缓冲液中并加热至沸腾，20min后自然冷却；用10％山羊血清进行封闭后加入500倍稀释的兔抗Eakr抗血清37℃温育2h，用TBST洗涤3次，加入HRP标记的山羊抗兔IgG二抗，37℃孵育30min，DAB显色；苏木精复染、脱水透明及树胶封片。阴性对照以阴性血清代替一抗；
步骤(6)其他鱼类Eakr同源蛋白的Western blot分析：
采用动物组织总蛋白提取试剂盒提取草鱼、黄颡、团头鲂、乌鳢和鲫鱼肝脏总蛋白，将50μg不同种鱼来源的肝脏总蛋白进行常规SDS-PAGE电泳及转膜；用自制的兔抗Eakr血清进行1：500倍稀释后作为一抗，用HRP标记的羊抗兔IgG为二抗，Western blot检测其他鱼类是否存在Eakr同源蛋白；
步骤(7)多克隆抗体的效价测定：
以纯化的重组蛋白为包被抗原，以第一次免疫前采集的兔血清为阴性对照，将兔抗Eakr抗体进行1：200至1：25600倍比稀释，以HRP标记的羊抗兔IgG为二抗，以TMB为底物显色液，进行间接ELISA分析；以兔抗Eakr抗体OD 450nm值与阴性对照血清OD450nm值的比值≥2时为阳性作为判定标准，制备的兔抗Eakr抗体最大稀释度作为该抗体的有效效价。
本发明与现有技术相比具有如下有益效果：
1)本发明采用分子生物学和基因工程的方法，构建了黄鳝Eakr基因的重组表达载体，诱导表达，亲和层析获得纯化的重组表达蛋白。
2)本发明的方法制备的黄鳝Eakr多克隆抗体具有很强的特异性，可用于免疫组化、免疫印迹等实验要求，建立体外免疫分析，为深入研究黄鳝Eakr的功能提供一个重要工具。
3)本发明制备的黄鳝Eakr多克隆抗体，可用于黄鳝免疫荧光染色激光共聚焦显微观察。
4)本发明制备的黄鳝Eakr多克隆抗体，还可用于其他鱼类Eakr的免疫检测。
附图说明
图1为黄鳝Eakr基因小量表达和纯化重组蛋白SDS-PAGE电泳图；
图2为黄鳝Eakr重组蛋白的Western blot检测图；
图3为以兔抗黄鳝Eakr血清为一抗的黄鳝各组织蛋白的Western blot检测图；
图4为黄鳝小肠，肝胰组织与脾脏的抗血清免疫组化分析对比组图；
图5为部分其他鱼类醛酮还原酶同源蛋白的Western blot检测图；
图6为兔抗黄鳝Eakr血清的效价测定结果示意图。
具体实施方式
下面详细描述本发明的实施例。下面通过参考附图描述的实施例是示例性的，仅用于解释本发明，而不能理解为对本发明的限制。
本发明实施方式提供一种黄鳝Eakr多克隆抗体的制备方法，包括以下步骤：
步骤1黄鳝Eakr基因的克隆：
按照Trizol试剂说明书提取黄鳝总RNA，利用M-MLV反转录酶合成cDNA，cDNA第一链的合成根据Smart RACE试剂盒说明书进行，于﹣80℃低温保存。根据GenBank数据库已报道的AKR基因序列进行同源比对，设计特异性引物AKR-F(5’-ACATCCAGACCAGCGACCTGCA-3’)和引物oligod(T)(5’-TGCCGAATCTTTTTTTTTTTTTAGC-3’)。以黄鳝cDNA第一链为模板，进行PCR扩增，获得黄鳝Eakr基因cDNA片段；特异性引物AKR-F与oligod(T)的核苷酸序列分别如SEQ ID NO：3和SEQ ID NO：4所示。
PCR反应体系(25μL)
PCR反应程序：
根据所得黄鳝Eakr基因cDNA片段设计特异性引物AKR-GSP5(5’-GTATCTCTTGTCGCTATAGTCCCACCAGTG-3’)和AKR-GSP3(5’-TGTGGGCTGCAGGCTAGGTGTCGC-3’)，特异性引物AKR-GSP5与AKR-GSP3的核苷酸序列分别如SEQ ID NO：5和SEQ ID NO：6所示。
按照Smart RACE试剂盒说明书进行5’RACE和3’RACE扩增，获得黄鳝Eakr基因cDNA片段的5’末端和3’末端。将黄鳝Eakr基因的5’末端和3’末端进行拼接，获得其cDNA全长序列。黄鳝Eakr基因的核苷酸和氨基酸序列分别如SEQ ID NO：1和SEQ ID NO：2所示。
5’RACE-PCR扩增的反应体系为(50μL)：
| 组成 | 体积(μL) |
| PCR-grade water | 35 |
| 10×BD Advantage buffer 2PCR buffer | 5 |
| dNTP Mix(10mM) | 1 |
| μPM | 5 |
| GSP | 1 |
| 5’-RACE-Ready cDNA | 2 |
| 150×BD Advantage 2Polymerase Mix | 1 |
3’RACE-PCR扩增的反应体系为(50μL)：
| 组成 | 体积(μL) |
| PCR-grade water | 35 |
| 10×BD Advantage buffer 2PCR buffer | 5 |
| dNTP Mix(10mM) | 1 |
| μPM | 5 |
| GSP | 1 |
| 3’-RACE-Ready cDNA | 2 |
| 150×BD Advantage 2Polymerase Mix | 1 |
反应条件为：
步骤2黄鳝Eakr基因重组表达载体的构建：
根据黄鳝Eakr基因的cDNA序列，设计一对特异性引物re-ex-F(5’-GCCGAATTCATGCCTGTGGTTCCCAAAG-3’)和re-ex-R(5’-CCGAAGCTTTTAGCTCCTGTACTCATCGC-3’)，分别在上、下游引物添加了EcoR I和Hind III酶切位点。以黄鳝cDNA为模板，采用引物re-ex-F和re-ex-R扩增黄鳝Eakr基因的成熟肽片段。特异性引物re-ex-F与re-ex-R的核苷酸序列分别如SEQ ID NO：7和SEQ ID NO：8所示。
PCR反应体系(25μL)：
反应条件为：
将PCR产物与重组表达载体pET-28a同时进行EcoR I和Hind III双酶切，分别用DNA凝胶回收试剂盒纯化酶切产物，经T4DNA连接酶连接后转化大肠杆菌DH5α，在含有卡那霉素(50μg/mL)的平板上进行抗性筛选。挑取阳性克隆接种到LB液体培养基中进行培养，37℃、220r/m，培养8h后用质粒提取试剂盒提取质粒，采用质粒PCR和双酶切的方法验证重组质粒。已成功构建重组表达载体，命名为pET-Eakr。
步骤3黄鳝Eakr重组蛋白的表达和纯化：
黄鳝Eakr重组蛋白的诱导表达：取1ng重组表达载体pET-Eakr转化大肠杆菌BL21(DE3)，涂布于含50μg/mL的卡那霉素平板上进行抗性筛选，培养温度为37℃，培养时间为过夜培养。筛选结束后转至LB培养基(含卡那霉素50μg/mL)中培养，培养条件为37℃，200r/min。菌体进入指数生长期后加添加IPTG至终浓度0.1mmol/L，进行IPTG诱导培养3小时后离心收集菌体沉淀，超声波破碎，加入5×上样缓冲液进行12％常规SDS-PAGE电泳检测重组蛋白的表达情况，重组蛋白分子量为37.7kDa。
黄鳝Eakr重组蛋白的纯化：将上述经过IPTG诱导的菌液按照1：100的比例接种到500mL LB培养基(含卡那霉素50μg/mL)中进行扩大培养，培养条件为37℃，200r/min培养时间为过夜培养。培养结束后，离心收集菌体沉淀，用50mmol/L的PBS(pH 8.0)悬浮菌液，进行超声波破碎并收集沉淀部分，用裂解缓冲液(6mol/L盐酸胍，10mmol/L咪唑，50mmol/L PBSpH＝8.0)洗涤3次。加入适量裂解缓冲液溶解，对上清液进行过滤；滤液用1mL的亲和层析镍柱His-Binding-Resin进行纯化；纯化后的蛋白用不同浓度的盐酸胍缓冲液进行透析复性，盐酸胍缓冲液浓度为6、3、1.5、0.5、0.1mol/L，用透析袋浓缩透析液；将收集到的黄鳝Eakr蛋白用BCA法测蛋白浓度，并用常规SDS-PAGE法，检测重组蛋白的条带大小，检测结果如图1所示。结果表明，黄鳝Eakr重组蛋白为单一条带，分子量为37.7kDa，其中M为DNA分子量标准；CK1为阴性对照；CK2为阳性对照；1为重组质粒pET-Eakr小量诱导产物；2为纯化的Eakr重组蛋白。
步骤4黄鳝Eakr重组蛋白多克隆抗体的制备：
将纯化的黄鳝Eakr重组蛋白和等体积的弗氏完全佐剂进行充分乳化；用背部皮下多点注射法，按0.2mg的免疫量对新西兰大白兔进行免疫，采集保存第一次免疫前新西兰大白兔的血清。用经弗氏不完全佐剂充分乳化的抗原进行加强免疫，每次间隔7天，共注射8次。末次免疫10天后，进行全血分离，收集兔抗黄鳝Eakr血清，加入30％甘油，于-20℃分装保存。将含有空白质粒的菌株总蛋白(阴性对照)、阳性菌株总蛋白(阳性对照)和纯化的蛋白进行SDS-PAGE电泳，然后将蛋白转至NC膜上。分别用武汉博士德生物有限公司制备的anti-His(1：2000倍稀释)及自制的兔抗血清1：500倍稀释后作为一抗，用HRP标记的羊抗兔IgG为二抗进行多抗的特异性检测。以第一次免疫前采集的兔血清为阴性对照。检测结果如图2所示，自制的兔抗Eakr抗血清也可以特异性地和纯化的蛋白产生一条特异条带；在和重组菌总蛋白反应时产生了一条分子量较小的条带，可能是过量表达的杂蛋白产生的非特异性反应导致；其中1为重组大肠杆菌BL21(DE3)总蛋白；2为纯化的黄鳝Eakr重组蛋白；a为商品anti-His作为一抗；b为用自制的兔抗Eakr血清作为一抗。
为进一步分析自制的抗血清的特异性，利用动物组织总蛋白提取试剂盒提取黄鳝肝胰组织、小肠、肌肉和脾脏4种组织的总蛋白。提取方法按照试剂盒说明书进行。总蛋白提取后取50μg各组织总蛋白进行SDS-PAGE电泳，然后将蛋白转至NC膜上。用制备的抗血清进行1：500倍稀释后作为一抗，用HRP标记的羊抗兔IgG为二抗进行多抗的特异性检测。检测结果如图3所示，来源于肝胰组织、小肠、肌肉和脾脏的总蛋白都可以和自制的兔抗Eakr血清产生特异性的反应，而且四种组织里带型和分子量大小均一致，为37.7kDa。
步骤5黄鳝Eakr蛋白的免疫组化分析：
采用石蜡包埋法包埋黄鳝的小肠、肝胰组织和脾脏，以6μm厚度进行切片。常规脱蜡后，用50μL的H2O2(3％)于37℃孵育10min，阻断内源过氧化物酶。将切片放入柠檬酸盐缓冲液中并加热至沸腾，20min后自然冷却。用10％山羊血清进行封闭后加入500倍稀释的兔抗黄鳝Eakr血清37℃温育2h，用TBST洗涤3次，加入HRP标记的山羊抗兔IgG二抗，37℃孵育30min，DAB显色。苏木精复染、脱水透明及树胶封片。阴性对照以实施例3中，多克隆抗体的制备步骤中的第一次免疫前新西兰大白兔的血清代替一抗。分析结果如图4所示，由免疫组化分析结果可知，黄鳝Eakr蛋白在黄鳝小肠中的整体分布较为分散，且在肠道内壁突出的绒毛根部分布相对较多，在肝胰组织和脾脏中分布较为集中，主要在肝胰组织和脾脏中的部分细胞中表达；其中图a、c、e为阴性对照效果图；b、d、f为经过兔抗黄鳝Eakr血清处理的效果图。而阴性对照未检出醛酮还原酶蛋白的表达。
步骤6其他鱼类Eakr同源蛋白的Western blot分析：
为验证部分其他鱼类体内是否存在醛酮还原酶同源蛋白，进行了Eakr同源蛋白的Western blot分析。具体过程如下：
用动物组织总蛋白提取试剂盒提取草鱼、黄颡、团头鲂、乌鳢和鲫鱼肝脏总蛋白，各取50μg所提取的蛋白进行常规SDS-PAGE纯化分离并转膜。以兔抗黄鳝Eakr血清进行1：500倍稀释后作为一抗，用HRP标记的羊抗兔IgG作为二抗进行Western blot检测，观察其是否有特异性反应。检测结果如图5所示，所检测的5种淡水鱼的肝脏组织总蛋白都可以特异性的和制备的多抗产生反应，且5种鱼的醛酮还原酶同源蛋白分子量比较一致，分子量大小为37.7kDa，说明鱼类的醛酮还原酶存在于不同物种；其中M为DNA分子量标准；1为草鱼；2为黄颡；3为团头鲂；4为乌鳢；5为鲫鱼。
步骤7多克隆抗体的效价测定：
为了对所获得的多克隆抗体的效价进行测定，具体过程如下：
以实施例4中，多克隆抗体的制备步骤中的第一次免疫前新西兰大白兔的血清为阴性对照，以最后一次免疫后收集的兔抗黄鳝Eakr血清在1：200至1：25600倍之间稀释作为一抗，以HRP标记的羊抗兔IgG为二抗，以TMB作为显色液，以兔抗黄鳝Eakr血清样品OD450nm值与阴性对照血清OD 450nm值的比值≥2时为阳性作为判定标准，以制备的兔抗黄鳝Eakr血清最大稀释度作为该抗体的有效效价。检测结果如图6所示，阳性血清的最高稀释度为1：12800，有效效价达到1：6400；其中纵坐标为OD 450nm吸光值，横坐标为兔抗Eakr血清的稀释倍数范围值；比较对象为阴性血清与兔抗血清。
本发明与现有技术相比具有如下有益效果：
1)本发明采用分子生物学和基因工程的方法，构建了黄鳝Eakr基因的重组表达载体，诱导表达，亲和层析获得纯化的重组表达蛋白。
2)本发明的方法制备的黄鳝Eakr多克隆抗体具有很强的特异性，可用于免疫组化、免疫印迹等实验要求，建立体外免疫分析，为深入研究黄鳝Eakr的功能提供一个重要工具。
3)本发明制备的黄鳝Eakr多克隆抗体，可用于黄鳝免疫荧光染色激光共聚焦显微观察。
4)本发明制备的黄鳝Eakr多克隆抗体，还可用于其他鱼类Eakr的免疫检测。
尽管已经示出和描述了本发明的实施例，本领域的普通技术人员可以理解：在不脱离本发明的原理和宗旨的情况下可以对这些实施例进行多种变化、修改、替换和变型，本发明的范围由权利要求及其等同物限定。
<110> 长江大学
<120>一种黄鳝醛酮还原酶多克隆抗体的制备方法
<160> 8
<210> 1
<211> 1320
<212> DNA13> 黄鳝（Monopterus albus）
<400> 1
acgttagtct gctgctgttg gttcgagcac agcttgtaag gaggacaaac ttctttccca 60
gcccttgtag caggatgcct gtggttccca aagtgacact gcctgggggt ctggagatct 120
gtcgggtcct gaatgggatg tggcaggtgt ctggtaagca cggggcagtg gatcgagcca 180
aagcagttga ggccatgcag gcctatgtag atgctggtct gaccacattc gacatggcag 240
acatttatgg gcctgcagag gaaatatttg ggcagttcaa cagcaagctg aagtcctctg 300
gagatgctgc accagctgtg cagagtctga ccaaatacgt tccacaacca ggaccaatgg 360
accgcaaggt ggtggagaag gcaatacagc tctccatgac tcggatgcag gtggacagtc 420
tggactgtgt tcagtttcac tggtgggact atagcgacaa gagatacctg gaggcactgg 480
gacacctgtt tgatatgcaa caggagggac tcatacgtga gctttccctc actaacttcg 540
acacacagag actggaggag atcagcaaca gaggcatccg catttccagc aaccaggttc 600
agtactctgt gattgaccag cgacctgcag tgaagatgga acagttctgt ctggccaatg 660
acatccagct cctcacttat ggcactctgg ctggcggtct gctcacagag cgctatctgg 720
gcaaggcgga gccaaactcc aaggcggagc tctatactgc ctccctctca agctacaaga 780
agatgatcga cacttggggt ggctggagcc ttttccagga cctacttgtt actttggagg 840
ctgttgccag gagacacaac tgccccatgg cctgtgtcgc aacgcgttac gtgctggacc 900
gccctgcagt gggcggggtc attgtgggct gcaggctagg tgtcgcaaat gcggggcagc 960
acatcggtga cagtctgcat agctgcagcc ctgacctgcg gttgacagcg gaggatctcg 1020
ctgctatcaa agctgtgaca cagcgctcca gggacctcat ggtgtggatt ggggactgcg 1080
gcgatgagta caggagctaa aggcagaagg ggtgaggaat agaggcagag taaagggaaa 1140
catgcagcag tcacacatgc aacagaataa aatgcagctg ctgctgaaaa tcatttcaat 1200
cctccatttc atttttaatt gaatcaaatt agtttttgca taaagtctgt agctgttcct 1260
ctgttacaca ctataaagaa ccaacagcaa attgtaaaaa aaaaaaaaaa aaaaaaaaaa 1320
<210> 2
<211> 341
<212> PRT
<213>黄鳝（Monopterus albus）
<400> 2
Met Pro Val Val Pro Lys Val Thr Leu Pro Gly Gly Leu Glu Ile Cys
1 5 10 15
Arg Val Leu Asn Gly Met Trp Gln Val Ser Gly Lys His Gly Ala Val
20 25 30
Asp Arg Ala Lys Ala Val Glu Ala Met Gln Ala Tyr Val Asp Ala Gly
35 40 45
Leu Thr Thr Phe Asp Met Ala Asp Ile Tyr Gly Pro Ala Glu Glu Ile
50 55 60
Phe Gly Gln Phe Asn Ser Lys Leu Lys Ser Ser Gly Asp Ala Ala Pro
65 70 75 80
Ala Val Gln Ser Leu Thr Lys Tyr Val Pro Gln Pro Gly Pro Met Asp
85 90 95
Arg Lys Val Val Glu Lys Ala Ile Gln Leu Ser Met Thr Arg Met Gln
100 105 110
Val Asp Ser Leu Asp Cys Val Gln Phe His Trp Trp Asp Tyr Ser Asp
115 120 125
Lys Arg Tyr Leu Glu Ala Leu Gly His Leu Phe Asp Met Gln Gln Glu
130 135 140
Gly Leu Ile Arg Glu Leu Ser Leu Thr Asn Phe Asp Thr Gln Arg Leu
145 150 155 160
Glu Glu Ile Ser Asn Arg Gly Ile Arg Ile Ser Ser Asn Gln Val Gln
165 170 175
Tyr Ser Val Ile Asp Gln Arg Pro Ala Val Lys Met Glu Gln Phe Cys
180 185 190
Leu Ala Asn Asp Ile Gln Leu Leu Thr Tyr Gly Thr Leu Ala Gly Gly
195 200 205
Leu Leu Thr Glu Arg Tyr Leu Gly Lys Ala Glu Pro Asn Ser Lys Ala
210 215 220
Glu Leu Tyr Thr Ala Ser Leu Ser Ser Tyr Lys Lys Met Ile Asp Thr
225 230 235 240
Trp Gly Gly Trp Ser Leu Phe Gln Asp Leu Leu Val Thr Leu Glu Ala
245 250 255
Val Ala Arg Arg His Asn Cys Pro Met Ala Cys Val Ala Thr Arg Tyr
260 265 270
Val Leu Asp Arg Pro Ala Val Gly Gly Val Ile Val Gly Cys Arg Leu
275 280 285
Gly Val Ala Asn Ala Gly Gln His Ile Gly Asp Ser Leu His Ser Cys
290 295 300
Ser Pro Asp Leu Arg Leu Thr Ala Glu Asp Leu Ala Ala Ile Lys Ala
305 310 315 320
Val Thr Gln Arg Ser Arg Asp Leu Met Val Trp Ile Gly Asp Cys Gly
325 330 335
Asp Glu Tyr Arg Ser
340
<210> 3
<211> 22
<212> DNA
<213> 人工序列
<400> 3
acatccagac cagcgacctg ca 22
<210> 4
<211> 25
<212> DNA
<213> 人工序列
<400> 4
tgccgaatct tttttttttt ttagc 25
<210> 5
<211> 30
<212> DNA
<213> 人工序列
<400> 5
gtatctcttg tcgctatagt cccaccagtg 30
<210> 6
<211> 24
<212> DNA
<213> 人工序列
<400> 6
tgtgggctgc aggctaggtg tcgc 24
<210> 7
<211> 28
<212> DNA
<213> 人工序列
<400> 7
gccgaattca tgcctgtggt tcccaaag 28
<210> 8
<211> 29
<212> DNA
<213> 人工序列
<400> 8
ccgaagcttt tagctcctgt actcatcgc 29
Claims (7)
1.一种黄鳝醛酮还原酶多克隆抗体的制备方法，其特征在于,包括以下步骤：
步骤(1)黄鳝Eakr基因的克隆：
提取黄鳝总RNA，用M-MLV反转录酶合成cDNA第一链，于-80℃保存；根据GenBank数据库中已报道的AKR基因序列，设计第一对特异性引物，进行PCR扩增，获得黄鳝Eakr基因cDNA片段；进行5’RACE-PCR和3’RACE-PCR扩增，获得黄鳝Eakr基因cDNA片段的5’末端和3’末端，拼接获得黄鳝Eakr基因全长cDNA序列；黄鳝Eakr基因的核苷酸和氨基酸序列分别如SEQ IDNO：1和SEQ ID NO：2所示；
步骤(2)黄鳝Eakr基因重组表达载体的构建：
根据黄鳝Eakr基因全长cDNA序列，设计第二对特异性引物re-ex-F和re-ex-R，分别在上、下游引物添加了EcoR I和Hind III酶切位点；以黄鳝cDNA为模板，re-ex-F和re-ex-R为引物，进行PCR扩增；将PCR产物和pET-28a(+)表达载体进行双酶切，回收纯化，连接转化大肠杆菌DH5α，获得重组表达载体pET-Eakr；
步骤(3)黄鳝Eakr重组蛋白的诱导表达和纯化：
将步骤(2)得到的重组表达载体pET-Eakr转化大肠杆菌BL21(DE3)，在含有50mg/L卡那霉素的LB培养液中培养至OD值为0.6时，加入IPTG进行诱导；离心收集菌体，超声破碎，然后加入5×上样缓冲液进行12％SDS-PAGE电泳检测目标蛋白的表达情况；
以1％的接种量将能表达重组蛋白的BL21添加到含有50μg/mL卡那霉素的LB培养基中，大量培养，诱导表达；将诱导好的菌液离心，收集菌体超声波破碎，再离心收集沉淀；将沉淀用平衡缓冲液溶解，收集上清，上样、结合、洗脱、透析，获得纯化的黄鳝Eakr重组蛋白；SDS-PAGE电泳检测纯化的黄鳝Eakr重组蛋白；
步骤(4)多克隆抗体的制备及Western blot分析：
将步骤(3)纯化的黄鳝pET-Eakr重组蛋白作为抗原，对新西兰大白兔进行8次免疫，按体积比1：1的比例加入弗氏完全佐剂，采用背部皮下多点注射；加强免疫选用弗氏不完全佐剂；末次免疫10天后，进行全血分离，收集兔抗血清，纯化得到黄鳝Eakr多克隆抗体。
2.如权利要求1所述的黄鳝醛酮还原酶多克隆抗体的制备方法，其特征在于，步骤(1)中，所述第一对特异性引物为：
引物名称为AKR-F，引物序列为：5’-ACATCCAGACCAGCGACCTGCA-3’；
引物名称为oligod(T)，引物序列为：
5’-TGCCGAATCTTTTTTTTTTTTTAGC-3’。
3.如权利要求1所述的黄鳝醛酮还原酶多克隆抗体的制备方法，其特征在于，步骤(1)中，设计第一对特异性引物，进行PCR扩增时，PCR扩增的反应体系为，总体积为25μL时：
反应条件为：
4.如权利要求1所述的黄鳝醛酮还原酶多克隆抗体的制备方法，其特征在于，步骤(2)中，所述第二对特异性引物为：
上游引物名称为re-ex-F，
引物序列为：5’-GCCGAATTCATGCCTGTGGTTCCCAAAG-3’；
下游引物名称为re-ex-R，
引物序列为：5’-CCGAAGCTTTTAGCTCCTGTACTCATCGC-3’；
任选地，PCR扩增的反应体系为，总体积为25μL时：
反应条件为：
5.如权利要求1所述的黄鳝醛酮还原酶多克隆抗体的制备方法，其特征在于，步骤(3)中，IPTG进行诱导时，IPTG的添加时间点为大肠杆菌BL21(DE3)进入指数生长期时，IPTG的终浓度为0.05mol/L，IPTG的诱导时间为3小时；所述裂解缓冲液组分为：6mol/L盐酸胍，10mmol/L咪唑，50mmol/L PBS。
6.如权利要求1所述的黄鳝醛酮还原酶多克隆抗体的制备方法，其特征在于，步骤(3)中，黄鳝Eakr重组蛋白的分子量为37.7kDa。
7.如权利要求1所述的黄鳝醛酮还原酶多克隆抗体的制备方法，其特征在于，步骤(4)中所述免疫的免疫量为0.2mg。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611152038.6A CN106478822B (zh) | 2016-12-14 | 2016-12-14 | 一种黄鳝醛酮还原酶多克隆抗体的制备方法 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611152038.6A CN106478822B (zh) | 2016-12-14 | 2016-12-14 | 一种黄鳝醛酮还原酶多克隆抗体的制备方法 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106478822A CN106478822A (zh) | 2017-03-08 |
| CN106478822B true CN106478822B (zh) | 2019-06-28 |
Family
ID=58284934
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201611152038.6A Active CN106478822B (zh) | 2016-12-14 | 2016-12-14 | 一种黄鳝醛酮还原酶多克隆抗体的制备方法 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106478822B (zh) |
Families Citing this family (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108192879B (zh) * | 2018-01-12 | 2021-04-13 | 长江大学 | 一种黄鳝醛酮还原酶突变蛋白的制备及应用 |
| CN110372794A (zh) * | 2019-06-04 | 2019-10-25 | 河南师范大学 | 中华鳖类固醇生成因子-1多克隆抗体的制备方法及应用 |
| CN111304177B (zh) * | 2020-02-21 | 2022-05-24 | 长江大学 | 一种重组蛋白swHO1的制备方法及应用 |
| CN112552405A (zh) * | 2021-01-06 | 2021-03-26 | 山东省淡水渔业研究院（山东省淡水渔业监测中心） | 抗草鱼pIgR多克隆抗体的制备方法及其应用 |
| CN113121691B (zh) * | 2021-04-13 | 2022-04-26 | 河南师范大学 | 鲤Toll样受体2多克隆抗体的制备方法及应用 |
| CN113564137B (zh) * | 2021-08-02 | 2023-04-25 | 长江大学 | 一种黄颡鱼醛还原酶蛋白及其制备方法和应用 |
| CN114106142B (zh) * | 2021-11-03 | 2024-05-14 | 中山大学 | 一种黄鳝生长催乳素抗血清及其制备方法与应用 |
| CN115806592B (zh) * | 2022-11-04 | 2023-06-09 | 山东省淡水渔业研究院（山东省淡水渔业监测中心） | 一种抗乌鳢pIgR抗体及其制备方法和应用 |
| CN120989034A (zh) * | 2025-10-27 | 2025-11-21 | 浙江省农业科学院 | 一种小黄鱼syvn1多克隆抗体及其制备方法、应用 |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8114606B2 (en) * | 2007-02-16 | 2012-02-14 | The Board Of Trustees Of Southern Illinois University | ARL-1 specific antibodies |
| EP2585488A2 (en) * | 2010-06-24 | 2013-05-01 | Modpro AB | Novel biomarkers of liver cancer |
| CN104650234B (zh) * | 2014-10-27 | 2018-07-06 | 湖南莱拓福生物科技有限公司 | 抗akr1b10蛋白单克隆抗体及其应用 |
| CN105779600A (zh) * | 2016-04-06 | 2016-07-20 | 浙江大学 | 一种akr1c1蛋白的应用 |
-
2016
- 2016-12-14 CN CN201611152038.6A patent/CN106478822B/zh active Active
Non-Patent Citations (2)
| Title |
|---|
| "PREDICTED: Lates calcarifer L-glyceraldehyde 3-phosphate reductase-like (LOC108891670), mRNA NCBI Reference Sequence: XM_018688927.1 ACCESSION NO XM_018688927";author;《genbank database》;20161014;FEATURES和ORIGIN * |
| "PREDICTED: aflatoxin B1 aldehyde reductase member 2-like [Neolamprologus brichardi]NCBI Reference Sequence: XP_006785476.1 ACCESSION NO:XP_006785476";AUTHOR;《GENBANK DATABASE》;20140210;FEATURES和ORIGIN * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106478822A (zh) | 2017-03-08 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106478822B (zh) | 一种黄鳝醛酮还原酶多克隆抗体的制备方法 | |
| Mamuti et al. | Echinococcus multilocularis: developmental stage-specific expression of Antigen B 8-kDa-subunits | |
| CN102746382B (zh) | 心脏型脂肪酸结合蛋白b细胞表位肽及其抗体和应用 | |
| Barderas et al. | Cloning, expression, and clinical significance of the major allergen from ash pollen, Fra e 1 | |
| Yokoyama et al. | Experimental hepatitis induced by ethanol after immunization with acetaldehyde adducts | |
| Wang et al. | A multigene family of Heteractis magnificalysins (HMgs) | |
| CN113150137A (zh) | 一种ndm-1单克隆抗体的制备方法及其应用 | |
| KR20230167444A (ko) | 알레르기의 항원 및 그 에피토프 | |
| CN101985475A (zh) | 新型分泌肽inm02的多克隆抗体及其制备方法和应用 | |
| TW200846366A (en) | Agent for suppressing rejection in organ transplantation comprising anti-hmgb-1 antibody | |
| Jiang et al. | CD4-1 and CD8α T lymphocytes subsets in spotted sea bass (Lateolabrax maculatus) and comparison on antigenicity of T lymphocytes subsets in other three marine fish species | |
| CN105801698A (zh) | 牛源vcam-1多克隆抗体的制备方法及应用 | |
| Sun et al. | Host protein EPCAM interacting with EtMIC8-EGF is essential for attachment and invasion of Eimeria tenella in chickens | |
| Nfon et al. | Langerhans cells in porcine skin | |
| CN106480074B (zh) | 一种黄鳝醛酮还原酶基因及其体外表达方法 | |
| CN109142738A (zh) | Ecm1作为血清学检测肝纤维化的标志物及其应用 | |
| CN107298697B (zh) | 一种人pd-l1蛋白y123位点磷酸化抗体及其制备方法和应用 | |
| Li et al. | Immobilization antigens provide orange-spotted grouper (Epinephelus coioides) protection against Cryptocaryon irritans infection | |
| CN112375769A (zh) | 一种葡萄牙牡蛎致敏蛋白ak的编码基因及其应用 | |
| Lv et al. | Characterization of a gene encoding prohibitin in silkworm, Bombyx mori | |
| WO2011074651A1 (ja) | シスタチンＣ、β２ミクログロブリン、α１ミクログロブリンおよびそれらの遺伝子、抗体、ならびに、ネコ腎症の診断用キット、診断方法 | |
| CN111363031B (zh) | BNIP3的pSer131多克隆抗体及其制备方法和应用 | |
| CN109295081A (zh) | 一种LDLR-Lamp2b融合基因、表达载体及其应用 | |
| Alvite et al. | Two novel Mesocestoides vogae fatty acid binding proteins–functional and evolutionary implications | |
| JP4041913B2 (ja) | 抗腫瘍タンパク質およびその遺伝子 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | 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 |