CN106854649B - The aptamer C204 and its screening technique of staphylococcus aureus enterotoxin C 2 and application - Google Patents
The aptamer C204 and its screening technique of staphylococcus aureus enterotoxin C 2 and application Download PDFInfo
- Publication number
- CN106854649B CN106854649B CN201611170854.XA CN201611170854A CN106854649B CN 106854649 B CN106854649 B CN 106854649B CN 201611170854 A CN201611170854 A CN 201611170854A CN 106854649 B CN106854649 B CN 106854649B
- Authority
- CN
- China
- Prior art keywords
- staphylococcus aureus
- aptamer
- aureus enterotoxin
- enterotoxin
- magnetic bead
- 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
- 241000191967 Staphylococcus aureus Species 0.000 title claims abstract description 95
- 231100000655 enterotoxin Toxicity 0.000 title claims abstract description 93
- 239000000147 enterotoxin Substances 0.000 title claims abstract description 85
- 101710146739 Enterotoxin Proteins 0.000 title claims abstract description 84
- 108091023037 Aptamer Proteins 0.000 title claims description 64
- 238000000034 method Methods 0.000 title abstract description 30
- 238000012216 screening Methods 0.000 title abstract description 24
- 238000000926 separation method Methods 0.000 claims description 5
- JWBWJOKTZVXSRT-DWQAGKKUSA-N 5-[(3as,4s,6ar)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]-2-aminopentanoic acid Chemical compound N1C(=O)N[C@@H]2[C@H](CCCC(N)C(O)=O)SC[C@@H]21 JWBWJOKTZVXSRT-DWQAGKKUSA-N 0.000 claims description 3
- LTMHDMANZUZIPE-AMTYYWEZSA-N Digoxin Natural products O([C@H]1[C@H](C)O[C@H](O[C@@H]2C[C@@H]3[C@@](C)([C@@H]4[C@H]([C@]5(O)[C@](C)([C@H](O)C4)[C@H](C4=CC(=O)OC4)CC5)CC3)CC2)C[C@@H]1O)[C@H]1O[C@H](C)[C@@H](O[C@H]2O[C@@H](C)[C@H](O)[C@@H](O)C2)[C@@H](O)C1 LTMHDMANZUZIPE-AMTYYWEZSA-N 0.000 claims description 3
- LTMHDMANZUZIPE-PUGKRICDSA-N digoxin Chemical compound C1[C@H](O)[C@H](O)[C@@H](C)O[C@H]1O[C@@H]1[C@@H](C)O[C@@H](O[C@@H]2[C@H](O[C@@H](O[C@@H]3C[C@@H]4[C@]([C@@H]5[C@H]([C@]6(CC[C@@H]([C@@]6(C)[C@H](O)C5)C=5COC(=O)C=5)O)CC4)(C)CC3)C[C@@H]2O)C)C[C@@H]1O LTMHDMANZUZIPE-PUGKRICDSA-N 0.000 claims description 3
- 229960005156 digoxin Drugs 0.000 claims description 3
- LTMHDMANZUZIPE-UHFFFAOYSA-N digoxine Natural products C1C(O)C(O)C(C)OC1OC1C(C)OC(OC2C(OC(OC3CC4C(C5C(C6(CCC(C6(C)C(O)C5)C=5COC(=O)C=5)O)CC4)(C)CC3)CC2O)C)CC1O LTMHDMANZUZIPE-UHFFFAOYSA-N 0.000 claims description 3
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 claims description 3
- 238000007385 chemical modification Methods 0.000 claims description 2
- 239000011324 bead Substances 0.000 abstract description 36
- 108020004414 DNA Proteins 0.000 abstract description 35
- 102000053602 DNA Human genes 0.000 abstract description 30
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 abstract description 7
- 108091008104 nucleic acid aptamers Proteins 0.000 abstract description 6
- 230000027455 binding Effects 0.000 abstract description 5
- 238000005516 engineering process Methods 0.000 abstract description 5
- 239000007790 solid phase Substances 0.000 abstract description 4
- 238000000338 in vitro Methods 0.000 abstract description 2
- 230000008685 targeting Effects 0.000 abstract 1
- FFRBMBIXVSCUFS-UHFFFAOYSA-N 2,4-dinitro-1-naphthol Chemical compound C1=CC=C2C(O)=C([N+]([O-])=O)C=C([N+]([O-])=O)C2=C1 FFRBMBIXVSCUFS-UHFFFAOYSA-N 0.000 description 9
- 238000004458 analytical method Methods 0.000 description 9
- 239000000243 solution Substances 0.000 description 8
- 101000686985 Mouse mammary tumor virus (strain C3H) Protein PR73 Proteins 0.000 description 7
- 238000012408 PCR amplification Methods 0.000 description 7
- 231100000617 superantigen Toxicity 0.000 description 7
- UBAVPHLFRVPRCI-CRBCFSCISA-N 2-[[(e)-2-carboxyethenyl]amino]-n-methoxy-2-oxoethanimine oxide Chemical compound CO\[N+]([O-])=C\C(=O)N\C=C\C(O)=O UBAVPHLFRVPRCI-CRBCFSCISA-N 0.000 description 6
- 238000010494 dissociation reaction Methods 0.000 description 6
- 230000005593 dissociations Effects 0.000 description 6
- 238000000746 purification Methods 0.000 description 6
- 241001478240 Coccus Species 0.000 description 5
- 101710084578 Short neurotoxin 1 Proteins 0.000 description 5
- 101710182532 Toxin a Proteins 0.000 description 5
- 241000219095 Vitis Species 0.000 description 5
- 235000009754 Vitis X bourquina Nutrition 0.000 description 5
- 235000012333 Vitis X labruscana Nutrition 0.000 description 5
- 235000014787 Vitis vinifera Nutrition 0.000 description 5
- 239000000872 buffer Substances 0.000 description 5
- 229940079593 drug Drugs 0.000 description 5
- 239000003814 drug Substances 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- 238000003786 synthesis reaction Methods 0.000 description 5
- 101000867232 Escherichia coli Heat-stable enterotoxin II Proteins 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 108010090804 Streptavidin Proteins 0.000 description 4
- 230000004913 activation Effects 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 201000010099 disease Diseases 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 239000012634 fragment Substances 0.000 description 4
- 210000000936 intestine Anatomy 0.000 description 4
- 239000003053 toxin Substances 0.000 description 4
- 231100000765 toxin Toxicity 0.000 description 4
- 206010040070 Septic Shock Diseases 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 238000004925 denaturation Methods 0.000 description 3
- 230000036425 denaturation Effects 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 208000015181 infectious disease Diseases 0.000 description 3
- 239000003446 ligand Substances 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 102000039446 nucleic acids Human genes 0.000 description 3
- 108020004707 nucleic acids Proteins 0.000 description 3
- 150000007523 nucleic acids Chemical class 0.000 description 3
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 3
- 230000035755 proliferation Effects 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
- 101150028074 2 gene Proteins 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 206010061218 Inflammation Diseases 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- 239000012980 RPMI-1640 medium Substances 0.000 description 2
- 210000001744 T-lymphocyte Anatomy 0.000 description 2
- 206010044248 Toxic shock syndrome Diseases 0.000 description 2
- 231100000650 Toxic shock syndrome Toxicity 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 230000008878 coupling Effects 0.000 description 2
- 238000010168 coupling process Methods 0.000 description 2
- 238000005859 coupling reaction Methods 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 230000004054 inflammatory process Effects 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 230000009871 nonspecific binding Effects 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 231100000614 poison Toxicity 0.000 description 2
- 239000002574 poison Substances 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 230000009870 specific binding Effects 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000002626 targeted therapy Methods 0.000 description 2
- 238000002560 therapeutic procedure Methods 0.000 description 2
- OTLLEIBWKHEHGU-UHFFFAOYSA-N 2-[5-[[5-(6-aminopurin-9-yl)-3,4-dihydroxyoxolan-2-yl]methoxy]-3,4-dihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-3,5-dihydroxy-4-phosphonooxyhexanedioic acid Chemical compound C1=NC=2C(N)=NC=NC=2N1C(C(C1O)O)OC1COC1C(CO)OC(OC(C(O)C(OP(O)(O)=O)C(O)C(O)=O)C(O)=O)C(O)C1O OTLLEIBWKHEHGU-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
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 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
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 102100031547 HLA class II histocompatibility antigen, DO alpha chain Human genes 0.000 description 1
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 1
- 101000866278 Homo sapiens HLA class II histocompatibility antigen, DO alpha chain Proteins 0.000 description 1
- 238000012404 In vitro experiment Methods 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 108010087230 Sincalide Proteins 0.000 description 1
- 206010041925 Staphylococcal infections Diseases 0.000 description 1
- 241000191940 Staphylococcus Species 0.000 description 1
- 230000005867 T cell response Effects 0.000 description 1
- 101710182223 Toxin B Proteins 0.000 description 1
- OKSSKVHGKYJNLL-LJRZAWCWSA-N [(3as,4r,9s,10as)-2,6-diamino-10,10-dihydroxy-9-sulfooxy-3a,4,8,9-tetrahydro-1h-pyrrolo[1,2-c]purin-4-yl]methoxycarbonylsulfamic acid Chemical compound OS(=O)(=O)NC(=O)OC[C@@H]1N=C(N)N2C[C@H](OS(O)(=O)=O)C(O)(O)[C@@]22N=C(N)N[C@H]21 OKSSKVHGKYJNLL-LJRZAWCWSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 238000001261 affinity purification Methods 0.000 description 1
- 238000003450 affinity purification method Methods 0.000 description 1
- 230000004523 agglutinating effect Effects 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 229940125644 antibody drug Drugs 0.000 description 1
- 239000003146 anticoagulant agent Substances 0.000 description 1
- 229940127219 anticoagulant drug Drugs 0.000 description 1
- 210000000612 antigen-presenting cell Anatomy 0.000 description 1
- 238000003149 assay kit Methods 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 239000012888 bovine serum Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 238000010523 cascade reaction Methods 0.000 description 1
- 238000010609 cell counting kit-8 assay Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000005034 decoration Methods 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000002405 diagnostic procedure Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 239000012636 effector Substances 0.000 description 1
- 235000014103 egg white Nutrition 0.000 description 1
- 210000000969 egg white Anatomy 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 238000012869 ethanol precipitation Methods 0.000 description 1
- 231100000776 exotoxin Toxicity 0.000 description 1
- 239000002095 exotoxin Substances 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 238000012215 gene cloning Methods 0.000 description 1
- 230000014509 gene expression Effects 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 229960002897 heparin Drugs 0.000 description 1
- 229920000669 heparin Polymers 0.000 description 1
- 238000012165 high-throughput sequencing Methods 0.000 description 1
- 239000005457 ice water Substances 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 239000002547 new drug Substances 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 239000002777 nucleoside Substances 0.000 description 1
- 125000003835 nucleoside group Chemical group 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 230000009465 prokaryotic expression Effects 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 238000000611 regression analysis Methods 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 238000004088 simulation Methods 0.000 description 1
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 230000009897 systematic effect Effects 0.000 description 1
- 239000013076 target substance Substances 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
- 239000011534 wash buffer Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- 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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
-
- 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
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/16—Aptamers
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/305—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Micrococcaceae (F)
- G01N2333/31—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Micrococcaceae (F) from Staphylococcus (G)
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- General Physics & Mathematics (AREA)
- Animal Behavior & Ethology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Food Science & Technology (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Plant Pathology (AREA)
- Pharmacology & Pharmacy (AREA)
- Cell Biology (AREA)
- Epidemiology (AREA)
- Pathology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Peptides Or Proteins (AREA)
Abstract
本发明涉及一种金黄色葡萄球菌肠毒素C2的核酸适配体C204及其筛选方法和应用，所述核酸适配体C204的序列为：AGGGCCGAGCTCACTTGTCTTAGTCCGACATGGTGCCTCCCTGCCCCAGAGCTCCGCCGGCACAATTGTGC；它的筛选方法为：基于核酸适配体的体外SELEX筛选技术，利用羧基磁珠作为固相介质，以金黄色葡萄球菌肠毒素C2为靶标，通过金黄色葡萄球菌肠毒素C2磁珠从ssDNA文库中筛选得到与金黄色葡萄球菌肠毒素C2特异性结合的核酸适配体；本发明的核酸适配体C204能高亲和力、高特异性地与金黄色葡萄球菌肠毒素C2结合。
The invention relates to a nucleic acid aptamer C204 of Staphylococcus aureus enterotoxin C2 and a screening method and application thereof. The sequence of the nucleic acid aptamer C204 is: AGGGCCGAGCTCACTTGTCTTAGTCCGACATGGTGCCTCCCTGCCCCAGAGCTCCGCCGGCACAATTGTGC; its screening method is: nucleic acid aptamer-based In vitro SELEX screening technology, using carboxyl magnetic beads as a solid-phase medium, targeting Staphylococcus aureus enterotoxin C2, and screening the ssDNA library with Staphylococcus aureus enterotoxin C2 magnetic beads to obtain a specific Sex-binding nucleic acid aptamer; the nucleic acid aptamer C204 of the present invention can bind to Staphylococcus aureus enterotoxin C2 with high affinity and high specificity.
Description
Technical field
The present invention relates to the aptamer C204 and its screening technique of a kind of staphylococcus aureus enterotoxin C 2 and answer
With.
Background technique
Staphylococcus aureus enterotoxin is the exotoxin with superantigen activity secreted by staphylococcus aureus.Gold
Staphylococcus aureus enterotoxin can be processed without antigen presenting cell, directly with the unrestricted combination of MHC class Ⅱmolecule, be formed
Compound in conjunction with the area β chain V of t lymphocyte antigen receptor, a large amount of activated T lymphocytes make its activation, proliferation, and release
The inflammatory cytokine of amplification quantity causes strong immune response, eventually leads to the damage of uncontrolled inflammation and multiple organ, causes poison
The diseases such as element shock.It is heat-resisting and only need very little dosage can further, since Staphylococcus aureus enterotoxin is relatively resistant to digest
Cause t cell response, Staphylococcus aureus enterotoxin is also by the super antigen drug as oncotherapy.
Staphylococcus aureus enterotoxin has multiple serotypes, is A type, Type B, C1 type, C2 type, C3 type, D type, E respectively
Type, G type, H-type, I type, M type, N-type, O-shaped and 1 type toxin of toxic shock syndrome, 2 type toxin of toxic shock syndrome etc.,
Middle staphylococcus aureus enterotoxin C 2 is not only related with staphylococcus aureus infections relating, while being also Staphylococcus aureus
Most super antigen drugs is applied in bacterium enterotoxin.
Detection staphylococcus aureus enterotoxin C 2 mainly uses immunological method, including precipitation reaction, agglutinating reaction,
ELISA, solid-phase RIA, biosensor etc., the knowledge that these methods use antibody to detect as staphylococcus aureus enterotoxin C 2
Other element, and the preparation of antibody is there are the period is long, complex steps, it is at high cost the deficiencies of place.In recent years, to detect golden yellow Portugal
Grape coccus Enteromycin C 2 gene is that the molecular biology method development of target is very fast, but the molecular biology methods such as PCR there are still
The technical problems such as false positive, false negative.Therefore, developing has more highly sensitive, economic, easy staphylococcus aureus intestines poison
Plain C2 new detecting technique, it is all significant for the diagnosis and treatment of Food Hygiene Surveillance, clinical infection of staphylococcus aureus etc..
Endotoxin Shock caused by staphylococcus aureus enterotoxin C 2 is treated, it is clinical to be propped up to the ill frequently with anti-inflammatory, fluid infusion etc.
Hold therapy.Current research discovery, inhibits the superantigen activity of staphylococcus aureus enterotoxin C 2, blocks inflammation in the initial stage
The generation of cascade reaction is the available strategy for treating staphylococcus aureus enterotoxin C 2 related disease.Based on this strategy, develop
Various polypeptide drugs, antibody drug, vaccine etc., it has also become staphylococcus aureus enterotoxin C 2 biotechnology new drug research
Hot spot.
The acquisition of staphylococcus aureus enterotoxin C 2 super antigen drug will be golden yellow frequently with the method for gene cloning
Aureus enterotoxin C 2 gene fragment clone in expression in escherichia coli and is purified into prokaryotic expression carrier.But this side
For purification step frequently with ion-exchange process or the affinity purification method of tape label, the former adsorbs selection poor specificity in method, after
Person's reagent cost is higher.
Aptamers are otherwise known as " synthetic antibody ", " chemical antibody ", and chemical nature is a single-stranded oligonucleotide molecule
(ssDNA or RNA) is folded into specific three dimensional structure in conjunction with target substance high-affinity and high specific.Aptamers are passed through
Phyletic evolution technology (the Systematic evolution of ligands by of index concentration ligand
Exponentialenrichment, SELEX) in-vitro screening process.Aptamer have high-affinity, high specific, can
External synthesis, can be changed by modification its function and pharmacokinetic properties, non-immunogenicity, it is economical the features such as.Based on above-mentioned
The aptamer drug of advantage exploitation can specific inhibition target function；Using aptamer as recognition component, can also open
Hair simplicity, accurately new detecting technique and efficient, economic affinity purification system.Therefore, high specific, Gao Qinhe are filtered out
The aptamer of power combination staphylococcus aureus enterotoxin C 2 has important scientific research, clinic and market value.
Summary of the invention
The purpose of the present invention is to provide a kind of Staphylococcus aureus enterotoxins with high specific and high-affinity
The aptamer C204 and its screening technique of C2 and application.
The purpose of the present invention is achieved through the following technical solutions: a kind of nucleic acid adaptation of staphylococcus aureus enterotoxin C 2
Body C204, its sequence are as follows:
AGGGCCGAGCTCACTTGTCTTAGTCCGACATGGTGCCTCC 40
CTGCCCCAGAGCTCCGCCGGCACAATTGTGC 71
The screening technique of the aptamer C204 of the staphylococcus aureus enterotoxin C 2 is based on aptamer
External SELEX screening technique, using carboxyl magnetic bead as solid-phase media, using staphylococcus aureus enterotoxin C 2 as target,
It is screened and is obtained and staphylococcus aureus enterotoxin C 2 spy from the library ssDNA by staphylococcus aureus enterotoxin C 2 magnetic bead
The aptamer that the opposite sex combines.
The application of the aptamer C204 of the staphylococcus aureus enterotoxin C 2, it is golden yellow in separation, purifying
Application in aureus enterotoxin C 2.
The application of the aptamer C204 of the staphylococcus aureus enterotoxin C 2, in analysis detection golden yellow Portugal
Non-disease diagnostic and therapeutic method application in grape coccus Enteromycin C 2.
The application of the aptamer C204 of the staphylococcus aureus enterotoxin C 2, in staphylococcus aureus intestines
Toxin C2 is the application in the targeted therapy of effector molecule.
The application of the aptamer C204 of the staphylococcus aureus enterotoxin C 2, in treatment Staphylococcus aureus
Cause the application in related syndrome in bacterium infection and in treatment staphylococcus aureus enterotoxin C 2.
For the prior art, the present invention has the advantages that
1. aptamer C204 is non-toxic, molecular weight is small, good penetrability, is readily synthesized and marks.
2. the synthesis cost of aptamer C204 is low compared with the cost of Antibody preparation, and the period is short, favorable reproducibility.
3. aptamer C204 can high-affinity, with high specificity in conjunction with staphylococcus aureus enterotoxin C 2,
Dissociation constant is 5.7 ± 0.26nM, and it does not have identification function to other homologous proteins.
4. separation, purifying of the aptamer C204 in staphylococcus aureus enterotoxin C 2, staphylococcus aureus
The relevant food safety detection of Enteromycin C 2, the diagnosing and treating of staphylococcus aureus enterotoxin C 2 related disease are golden yellow
The diagnosing and treating of staphy lococcus infection, targeted therapy that staphylococcus aureus enterotoxin C 2 mediates etc. have extensively in terms of fields
Wealthy application prospect and important science, society, economic value.
Detailed description of the invention
Fig. 1 is the biological information simulation drawing of aptamer C204 secondary structure.
The specificity figure that Fig. 2 is fluorescence Percentage bound experimental analysis aptamer C204.In Fig. 2, abscissa is analysis
Albumen, ordinate be fluorescence Percentage bound.
Fig. 3 is the dissociation of fluorescence Percentage bound experimental analysis aptamer C204 combination staphylococcus aureus enterotoxin C 2
Constant draws curve.Dissociation constant (Kd) is 5.7 ± 0.26nM.In Fig. 3, abscissa is DNA concentration (pM), and ordinate is glimmering
Light Percentage bound.
Fig. 4 is the PBMC proliferation that CCK-8 method measures that aptamer C204 induces staphylococcus aureus enterotoxin C 2
Active effect.
Specific embodiment
The content of present invention is described in detail with embodiment with reference to the accompanying drawings of the specification:
A kind of aptamer C204 of staphylococcus aureus enterotoxin C 2, its sequence are as follows:
AGGGCCGAGCTCACTTGTCTTAGTCCGACATGGTGCCTCC 40
CTGCCCCAGAGCTCCGCCGGCACAATTGTGC 71
The aptamer C204 of the staphylococcus aureus enterotoxin C 2, at 25 DEG C, 100mM Na+, 1mM Mg2+
Under conditions of, space structure is as follows:
The aptamer C204 of the staphylococcus aureus enterotoxin C 2, to the 5 ' of the aptamer C204
End or 3 ' ends carry out FITC, amino, biotin, digoxin chemical modification.
The aptamer C204 of the staphylococcus aureus enterotoxin C 2 cuts the aptamer C204
The obtained product of structure of modification of short or extension or number of base replacement carries out FITC, amino, biotin, digoxin chemistry and repairs
Decorations.
The screening technique of the aptamer C204 of the staphylococcus aureus enterotoxin C 2 is based on aptamer
External SELEX screening technique, using carboxyl magnetic bead as solid-phase media, using staphylococcus aureus enterotoxin C 2 as target,
It is screened and is obtained and staphylococcus aureus enterotoxin C 2 spy from the library ssDNA by staphylococcus aureus enterotoxin C 2 magnetic bead
The aptamer that the opposite sex combines.
The screening technique of the aptamer C204 of the staphylococcus aureus enterotoxin C 2, it includes following step
It is rapid:
(1) it screens the preparation in library: preparing ssDNA pool shown in following sequence:
5’-AGGGCCGAGCTCACTTGT-N35-CCGCCGGCACAATTGTGC-3'；
(2) staphylococcus aureus enterotoxin C 2 and the coupling of carboxyl magnetic bead are prepared into staphylococcus aureus enterotoxin C 2 magnetic
Pearl；
(3) library ssDNA is subjected to hot activation processing；
(4) will through after step (3) the library ssDNA and step (2) resulting staphylococcus aureus enterotoxin C 2 magnetic bead
It is incubated for；
(5) staphylococcus aureus enterotoxin C 2 magnetic bead of the Magnetic Isolation after step (4), washes away staphylococcus aureus
Enteromycin C 2 magnetic bead surfaces are unbonded, weak binding and non-specific binding ssDNA；Heat staphylococcus aureus enterotoxin C 2
Magnetic bead collects the ssDNA with the specific binding of staphylococcus aureus enterotoxin C 2 magnetic bead, i.e. ssDNA enriched library；
(6) PCR amplification: the resulting ssDNA enriched library of step (5) is subjected to PCR amplification, wherein used in PCR amplification
Primer are as follows:
Primer P1:5 '-FAM-AGGGCCGAGCTCACTTGT-3 '
Primer P2:5 '-Biotin-GCACAATTGTGCCGGCGG-3 '；
(7) purifying of PCR product: PCR product is purified using small fragment purification kit；It will after purification
DsDNA is incubated for Streptavidin MagneSphere, in conjunction with dsDNA Streptavidin MagneSphere is washed, after dsDNA unwinding, use
Magnetic frame separation, collects supernatant；Supernatant obtains the secondary library ssDNA for the next round of screening after ethanol precipitation；
(8) Cycle Screening: time by the secondary library ssDNA of the resulting FAM label of step (7), as next round screening
Grade library, and repeat the screening process of step (3)~(7).
Embodiment one: the screening of aptamer C204
The screening technique of the aptamer C204 of the staphylococcus aureus enterotoxin C 2, it includes following step
It is rapid:
(1) screen the preparation in library: design both ends fixed area is 18 nucleotide, intermediate random areas is 35 nucleosides
SsDNA pool (the 5 '-AGGGCCGAGCTCACTTGT-N of acid35- CCGCCGGCACAATTGTGC-3 '), and student on commission's work
The synthesis of bioengineering limited liability company.
(2) staphylococcus aureus enterotoxin C 2 and carboxyl magnetic bead are coupled: the staphylococcus aureus enterotoxin C 2 egg
White to be purchased from U.S. Toxin Technology company, the carboxyl magnetic bead and its coupling reagent are purchased from U.S. Bangs
Laboratories company, the specification that operation is provided referring to manufacturer；It is golden yellow that front and back is coupled by BCA method determination of protein concentration
The variation of protein concentration in color aureus enterotoxin C 2 solution, the efficiency that couples for being computed magnetic bead is 85%；By golden yellow Portugal
Grape coccus Enteromycin C 2 magnetic bead is scattered in PBS buffer solution, 4 DEG C of preservations.
(3) take 2nmol ssDNA pool be dissolved in 500 μ L selection buffer (50mM Tris-HCl, 100mM NaCl,
1mM MgCl2, 5mM KCl, pH7.4), then handled through hot activation.Wherein, the method for hot activation processing are as follows: 95 DEG C of denaturation
After 5min, it is immediately placed on ice bath 10min in ice-water bath, is subsequently placed at room temperature 10min.
(4) will through after step (3) the library ssDNA and step (2) resulting staphylococcus aureus enterotoxin C 2 magnetic bead
(staphylococcus aureus enterotoxin C 2 carrying capacity is 100ng) and yeast tRNA (mole be the library ssDNA 5 times) mixing are simultaneously
In incubation at room temperature 1h.
(5) staphylococcus aureus enterotoxin C 2 magnetic bead of the Magnetic Isolation after step (4), with the selection containing 0.2%BSA
Buffer washes away that staphylococcus aureus enterotoxin C 2 magnetic bead surfaces are unbonded, ssDNA of weak binding and non-specific binding；So
Afterwards by staphylococcus aureus enterotoxin C 2 magnetic bead with 200 μ L ddH2O is resuspended, and after 100 DEG C of hot bath 5min, is placed in magnetic frame
1-2min collects supernatant, obtains the ssDNA with the specific binding of staphylococcus aureus enterotoxin C 2 magnetic bead, i.e. ssDNA enrichment
Library.
(6) PCR amplification: the resulting ssDNA enriched library of step (5) is added in 1mL PCRmix；Vortex oscillation is mixed
After even, dispensed by every 50 μ L of pipe and carry out PCR amplification, amplification condition are as follows: after 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 30S, 58 DEG C are moved back
Fiery 30S, 72 DEG C of extension 30S, 15-25 circulation.
Wherein contain in 1mL PCRmix: 10 × PCR buffer, 100 μ L；3 μ L of pfu enzyme；dNTP 20μL；Primer P1:5 '-
Each 3 μ L of FAM-AGGGCCGAGCTCACTTGT-3 ' and primer P2:5 '-Biotin-GCACAATTGTGCCGGCGG-3 '；It is described to draw
The equal student on commission's work bioengineering limited liability company synthesis of object P1 and primer P2.
(7) purifying of PCR product: both ends indicate the PCR product of biotin and fluorophor FAM respectively, use small fragment
Purification Kit (the small fragment purification kit be purchased from Sheng Gong bioengineering limited liability company), will after purification
DsDNA and Streptavidin MagneSphere (being purchased from Invitrogen-Dynal company) use washing buffer in 37 DEG C of incubation 20min
(5mM Tris-HCl, pH7.5,1M NaCl, 500 μM of EDTA) washing in conjunction with dsDNA Streptavidin MagneSphere three times after, use
50 μ L NaOH solutions (0.1M) make dsDNA unwinding in 37 DEG C of incubation 30min；It is separated with magnetic frame, collects supernatant, supernatant is through second
Alcohol precipitating obtains the secondary library ssDNA of FAM label, and is dissolved in selection buffer, the secondary text as next round screening
Library.
(8) screening process carries out 9 wheels altogether.Since the second wheel, the dosage of secondary library is 50pmol.
Embodiment two: the analysis of aptamer C204 sequence:
(1) after 9 wheel screenings, the library ssDNA of enrichment is collected, and entrusts the upper gloomy promise biotechnology share of the Shanghai's style limited
Company analyzes library sequence using high throughput sequencing technologies, analytic process are as follows: PCR amplification enriched library, and plus survey
Sequence connector and the part Index；Purified library is selected by gel electrophoresis；Passed through using Agilent 2100Bioanalyzer
Agilent High Sensitivity DNA Kit is to library Quality Control；Utilize Quant-iT PicogGreen dsDNA
Assay Kit quantifies library；Using 500 platform of IlluminateNextSeq, bridge is carried out by template of single-stranded library
Formula PCR amplification, sequencing primer annealing, the sequencing of the side Bian Hecheng；And analysis is compared and is enriched with to sequencing result.
(2) using the analysis of the UNAFold network platform at 25 DEG C, 100mM Na+, 1mM Mg2+Under conditions of, aptamer
The secondary structure of C204 sequence.The secondary structure schematic diagram for analyzing aptamer C204 sequence is as shown in Figure 1.
Embodiment three: the specificity analysis of aptamer C204:
(1) the aptamer C204 of iii vitro chemical synthesis FAM label, and it is dissolved in selection buffer.
(2) referring to step (2) in embodiment one, by BSA, staphylococcus aureus toxin A, staphylococcus aureus intestines
Toxin B and Staphylococcal enterotoxin C1 couple preparation BSA magnetic bead, staphylococcus aureus intestines with carboxyl magnetic bead respectively
Toxin A magnetic bead, Staphylococcal enterotoxin B magnetic bead, Staphylococcal enterotoxin C1 magnetic bead and golden yellow grape
Coccus Enteromycin C 2 magnetic bead.Wherein, the BSA is purchased from Sigma company, the staphylococcus aureus toxin A, golden yellow Portugal
Grape coccus enterotoxin B and Staphylococcal enterotoxin C1 are purchased from Toxin Technology company, the U.S..
(3) take the 200 resulting aptamer C204 solution of μ L step (1) respectively with BSA magnetic bead made from step (2),
Staphylococcus aureus toxin A magnetic bead, Staphylococcal enterotoxin B magnetic bead, Staphylococcal enterotoxin C1 magnetic
Pearl and the mixing of staphylococcus aureus enterotoxin C 2 magnetic bead, are incubated at room temperature 1h in magazine, if blank magnetic bead is control.
(4) above-mentioned magnetic bead 3 times through step (3) are washed with 0.1%PBST, the aptamer in conjunction with above-mentioned magnetic bead,
100 DEG C of buffer are selected to boil 5min elution with 200 μ L.
(5) measure the fluorescence intensity of initial soln and eluent respectively using fluorescent quantitation instrument, calculate fluorescence Percentage bound=
(initial fluorescent intensity-elution fluorescence intensity)/initial fluorescent intensity × 100%, tentatively represents aptamer with calculated value
The Percentage bound of C204 and target molecule.
As shown in Fig. 2, the Percentage bound of aptamer C204 and staphylococcus aureus enterotoxin C 2 is all remarkably higher than it
With BSA, staphylococcus aureus toxin A, Staphylococcal enterotoxin B, Staphylococcal enterotoxin C1 knot
Conjunction rate shows that the combination of aptamer C204 and staphylococcus aureus enterotoxin C 2 has preferable specificity.
Example IV: the affinity analysis of aptamer C204
(1) take the FAM labeling nucleic acid aptamers C204 solution of various concentration respectively with staphylococcus aureus enterotoxin C 2
Magnetic bead mixing, is incubated at room temperature 1h in magazine.
(2) referring to the step (4) and step (5) in embodiment three, experiment obtains and calculates various concentration aptamer
The fluorescence Percentage bound of C204 solution and staphylococcus aureus enterotoxin C 2 magnetic bead.
(3) calculated value for utilizing fluorescence Percentage bound, draws aptamer C204 combination Staphylococcus aureus enterotoxin
The saturation binding curve of C2 calculates aptamer C204 combination Staphylococcus aureus enterotoxin by nonlinear regression analysis
The dissociation constant of C2.
As shown in figure 3, being computed aptamer C204 we obtain the saturation binding curve of aptamer C204
Dissociation constant be 5.7 ± 0.26nM, show the combination in conjunction with aptamer C204 and staphylococcus aureus enterotoxin C 2
Ability is strong, and dissociation constant is in nanomole rank.
Embodiment five: the superantigen activity of aptamer C204 inhibition staphylococcus aureus enterotoxin C 2
(1) aseptic aspiration healthy adult volunteer peripheral blood is diluted after anticoagulant heparin with equivalent RPMI1640 culture solution, is set
In on lymphocyte separation medium, it is single that acquisition human peripheral is separated using Conventional density gradients centrifugal process (2000rmp, 20min)
Nucleus (PBMCs).
(2) cell concentration is adjusted with the RPMI1640 culture solution containing 5% newborn bovine serum (NBS) and 5% Human autologous serum
It is 1~2 × 106/ mL spreads 96 orifice plates, every 100 μ L of hole.
(3) concentration that aptamer C204 is added in each group is followed successively by 0 μM, 10 μM respectively, and every hole is added final concentration of
The staphylococcus aureus enterotoxin C 2 of 250ng/mL, setting are not added staphylococcus aureus enterotoxin C 2, nucleic acid are also not added
The blank control group of aptamers C204.
(4) cell is placed in 37 DEG C, 5%CO2CO2It is cultivated in incubator, after culture for 24 hours, the CCK- of 10 μ L is added in every hole
8, continue to cultivate 4h.
(5) spectrophotometer detects every hole in the absorbance (OD) of 450nm.
As shown in figure 4, abscissa is the concentration of aptamer C204, ordinate is the OD value at 450nm.When nucleic acid is suitable
When ligand C204 is 10 μM, the proliferation water of staphylococcus aureus enterotoxin C 2 stimulation PBMCs is flat to be substantially reduced (P < 0.05),
The above results show that aptamer C204 in vitro experiment has the superantigen activity of staphylococcus aureus enterotoxin C 2
There is significantly inhibiting effect, is a kind of potential staphylococcus aureus enterotoxin C 2 inhibitor.
SEQUENCE LISTING
<110>Fuzhou General Hospital, Nanjing Military Area, PLA
<120>the aptamer C204 of staphylococcus aureus enterotoxin C 2 and its screening technique and application
<160> 3
<210> 1
<211> 71
<212> DNA
<213>artificial sequence
<400> 1
agggccgagc tcacttgtct tagtccgaca tggtgcctcc 40
ctgccccaga gctccgccgg cacaattgtg c 71
<210> 2
<211> 18
<212> DNA
<213>artificial sequence
<400> 2
agggccgagc tcacttgt 18
<210> 3
<211> 18
<212> DNA
<213>artificial sequence
<400> 3
gcacaattgt gccggcgg 18
Claims (3)
1. a kind of aptamer C204 of staphylococcus aureus enterotoxin C 2, it is characterised in that: its sequence such as SEQ ID
Shown in NO.1；And it is at 25 DEG C, 100mM Na+, 1mM Mg2+Under conditions of, space structure is as follows:
2. the aptamer C204 of staphylococcus aureus enterotoxin C 2 according to claim 1, it is characterised in that: right
5 ' the ends or 3 ' ends of the aptamer C204 carry out FITC, amino, biotin, digoxin chemical modification.
3. the aptamer C204 of staphylococcus aureus enterotoxin C 2 according to claim 1 or 2 is in separation, purifying
Application in staphylococcus aureus enterotoxin C 2.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611170854.XA CN106854649B (en) | 2016-12-16 | 2016-12-16 | The aptamer C204 and its screening technique of staphylococcus aureus enterotoxin C 2 and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611170854.XA CN106854649B (en) | 2016-12-16 | 2016-12-16 | The aptamer C204 and its screening technique of staphylococcus aureus enterotoxin C 2 and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106854649A CN106854649A (en) | 2017-06-16 |
| CN106854649B true CN106854649B (en) | 2019-05-21 |
Family
ID=59126010
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201611170854.XA Active CN106854649B (en) | 2016-12-16 | 2016-12-16 | The aptamer C204 and its screening technique of staphylococcus aureus enterotoxin C 2 and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106854649B (en) |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| IL158140A0 (en) * | 2003-09-25 | 2004-03-28 | Hadasit Med Res Service | Multiepitope polypeptides for cancer immunotherapy |
| CN103243101B (en) * | 2013-05-16 | 2015-01-14 | 江南大学 | One group of aptamers for specifically recognizing staphylococcus aureus enterotoxin C1 |
-
2016
- 2016-12-16 CN CN201611170854.XA patent/CN106854649B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN106854649A (en) | 2017-06-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP3143477B2 (en) | Method based on the use of bacteriophage for the detection of biological molecules in biological samples | |
| CN103224936B (en) | The aptamer of one group-specific identification staphylococcus aureus toxin A | |
| CN103013999B (en) | Oligonucleotides aptamer special for distinguishing fumonisin B1 | |
| CN105985962B (en) | The aptamer of the selectively targeted inflammatory synovial cell of rheumatoid arthrosis and its application | |
| CN106591315B (en) | The aptamer C202 and its screening technique of staphylococcus aureus enterotoxin C 2 and application | |
| CN102010866B (en) | An oligonucleotide aptamer that specifically recognizes Shigella | |
| CN103243101B (en) | One group of aptamers for specifically recognizing staphylococcus aureus enterotoxin C1 | |
| CN106701768B (en) | Nucleic acid aptamer C201 of Staphylococcus aureus enterotoxin C2 and its screening method and application | |
| CN106636105B (en) | Nucleic acid aptamer C203 of Staphylococcus aureus enterotoxin C2 and its screening method and application | |
| CN103014000B (en) | A group of oligonucleotide aptamers that specifically recognize Streptococcus agalactiae | |
| CN106854649B (en) | The aptamer C204 and its screening technique of staphylococcus aureus enterotoxin C 2 and application | |
| CN106801059B (en) | Nucleic acid aptamer C205 of Staphylococcus aureus enterotoxin C2 and its screening method and application | |
| CN105483133B (en) | - 1 aptamer T-7 of Staphylococcus aureus toxic shock toxin and its preparation method and application | |
| CN110819632B (en) | Aptamer for binding to trastuzumab | |
| CN102766632B (en) | Golgi apparatus membrane protein GP73 nucleic acid aptamer, as well as preparation method and application thereof | |
| CN111793629A (en) | Aptamer ETA01 of pseudomonas aeruginosa exotoxin A and application thereof | |
| CN110885829B (en) | Diphtheria toxin aptamer DT05 and application thereof | |
| CN100519759C (en) | A process for preparation of an agglutination reagent for rapid detection of typhoid | |
| CN110885828B (en) | Diphtheria toxin aptamer DT01 and application thereof | |
| CN110904112B (en) | Diphtheria toxin aptamer DT03 and application thereof | |
| CN110938631B (en) | Diphtheria toxin aptamer DT02 and application thereof | |
| CN110923239B (en) | Diphtheria toxin aptamer DT04 and application thereof | |
| CN114410640B (en) | Aptamer for detecting measles virus, kit and application | |
| CN115786350B (en) | Aptamer capable of specifically recognizing and combining peripheral blood T lymphocytes, complementary sequence and application thereof | |
| CN108396029A (en) | One group-specific identifies different growing stage Escherichia coli O 157:The oligonucleotides aptamers of H7 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| 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 |